Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics in response to cold

ABSTRACT

Methods and materials for modulating cold tolerance levels in plants are disclosed. For example, nucleic acids encoding cold tolerance-modulating polypeptides are disclosed as well as methods for using such nucleic acids to transform plant cells. Also disclosed are plants having increased cold tolerance levels and plant products produced from plants having increased cold tolerance levels.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING OR TABLE

The material in the accompanying sequence listing is hereby incorporatedby reference into this application. The accompanying file, named2008-12-11_(—)2750-1707WO1_Sequence Listing.txt was created on Dec. 11,2008 and is 850 KB. The file can be accessed using Microsoft Word on acomputer that uses Windows OS.

TECHNICAL FIELD

The present invention relates to methods and materials involved inmodulating cold tolerance in plants, including growth levels in plantsgrown under low or chilling temperature stress conditions (a.k.a. “coldstress”). For example, this invention provides plants having increasedgrowth rate, vegetative growth, seedling vigor and/or biomass under coldstress conditions as compared to wild-type plants grown under similarconditions, as well as materials and methods for making plants and plantproducts having increased growth levels under cold stress conditions.

BACKGROUND

Plants are constantly exposed to a variety of biotic (i.e. pathogeninfection and insect herbivory) and abiotic (i.e. high or lowtemperature, drought, flood and salinity) stresses. To survive thesechallenges to their sessile life, plants have developed elaboratemechanisms to perceive external signals and to manifest adaptiveresponses with proper physiological and morphological changes (Bohnertet al. 1995). Plants exposed to cold or chilling conditions typicallyhave low yields of biomass, seeds, fruit and other edible products. Theterm “chilling sensitivity” is used for the description of physiologicaland developmental damages in the plant caused by low, but abovefreezing, temperatures. Important agricultural crop plants such as corn,soybean, rice and cotton have tropical ancestors that make them chillingsensitive. In some countries or agricultural regions of the worldchilling temperatures are a significant cause of crop losses and aprimary factor limiting the geographical range and growing season ofmany crop species. Another example is that chilling conditions can causesignificant concern in early spring planting of corn or canola. Poorgermination and reduced growth of chilling sensitive crops in the springresults in less ground coverage, more erosion and increased occurrenceof weeds leading to less nutrient supply for the crop.

Typically, chilling damage includes wilting, necrosis or ion leakagefrom cell membranes, especially calcium leakage, and decreased membranefluidity, which consequently impacts membrane dependent processes suchas: photosynthesis, protein synthesis, ATPase activity, uptake ofnitrogen, etc. (see Levitt J (1980) Chilling injury and resistance. InChilling, Freezing, and High Temperature Stresses: Responses of Plant toEnvironmental Stresses, Vol 1., T T Kozlowsky, ed, Academic Press, NewYork, pp 23-64; Graham and Patterson (1982) Annu Rev Plant Physiol 33:347-372; Guy (1990) Annu Rev Plant Physiol Plant Mol Biol 41: 187-223;and Nishida and Murata (1996) Annu Rev Plant Physiol Plant Mol Biol 47:541-568.). In addition, cold temperatures are often associated with wetconditions. The combination of cold and wet can result in hypoxic stresson the roots, causing an even more severe reduction of growth rate but,more critically, can be lethal to the plants, especially sensitive plantspecies such as corn and cotton.

Yet it has been observed that environmental factors, such as lowtemperature, can serve as triggers to induce cold acclimation processesallowing plants responding thereto to survive and thrive in lowtemperature environments. It would, therefore, be of great interest andimportance to be able to identify genes that regulate or confer improvedcold acclimation characteristics to enable one to create transformedplants (such as crop plants) with improved cold tolerancecharacteristics such as faster germination and/or growth and/or improvednitrogen uptake under cold conditions to improve survival or performanceunder low or chilling temperatures.

In the fields of agriculture and forestry, efforts are constantly beingmade to produce plants with an increased growth potential in order tofeed the ever-increasing world population and to guarantee the supply ofreproducible raw materials. This is done conventionally through plantbreeding. The breeding process is, however, both time-consuming andlabor-intensive. Furthermore, appropriate breeding programs must beperformed for each relevant plant species.

Progress has been made in part by the genetic manipulation of plants;that is by introducing and expressing recombinant nucleic acid moleculesin plants. Such approaches have the advantage of not usually beinglimited to one plant species, but instead being transferable among plantspecies. There is a need for generally applicable processes that improveforest or agricultural plant growth potential. Therefore, the presentinvention relates to a method for increasing growth potential,decreasing chilling damage, and/or increasing levels of cold acclimationin plants under low temperature, chilling or cold conditions,characterized by expression of recombinant DNA molecules stablyintegrated into the plant genome.

SUMMARY

The present invention provides methods and materials related to plantshaving modulated levels of cold tolerance. For example, this inventionprovides transgenic plants and plant cells having increased levels ofcold tolerance, nucleic acids (i.e. isolated polynucleotides),polypeptides encoded thereby used to generate transgenic plants andplant cells having increased levels of cold tolerance, and methods formaking plants and plant cells having increased levels of cold tolerance.Such plants and plant cells having increased cold tolerance will producebiomass under cold stress conditions that may be useful in producingbiomass for conversion to a liquid fuel or other chemicals, or may beuseful as a thermochemical fuel.

Methods of producing a plant and/or plant tissue are provided herein. Inone aspect, a method comprises growing a plant cell comprising anexogenous nucleic acid. The exogenous nucleic acid comprises aregulatory region operably linked to a nucleotide sequence encoding apolypeptide. The Hidden Markov Model (HMM) bit score of the amino acidsequence of the polypeptide is greater than about 130, 180, 650, or 315,using an HMM generated from the amino acid sequences depicted in one ofFIG. 1, 2, 3, 4, or 5, respectively. The plant and/or plant tissue has adifference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise the exogenous nucleic acid.

In another aspect, a method comprises growing a plant cell comprising anexogenous nucleic acid. The exogenous nucleic acid comprises aregulatory region operably linked to a nucleotide sequence encoding apolypeptide having 80 percent or greater sequence identity to an aminoacid sequence set forth in SEQ ID NOs: 2, 20, 74, or 93. A plant and/orplant tissue produced from the plant cell has a difference in the levelof cold tolerance as compared to the corresponding level of coldtolerance of a control plant that does not comprise the exogenousnucleic acid.

In another aspect, a method comprises growing a plant cell comprising anexogenous nucleic acid. The exogenous nucleic acid comprises aregulatory region operably linked to a nucleotide sequence having 80percent or greater sequence identity to a nucleotide sequence or at afragment thereof set forth in SEQ ID NO: 1, 19, 92, 97, or 111. A plantand/or plant tissue produced from the plant cell has a difference in thelevel of cold tolerance as compared to the corresponding level of coldtolerance of a control plant that does not comprise the exogenousnucleic acid.

Methods of modulating the level of cold tolerance in a plant areprovided herein. In one aspect, a method comprises introducing into aplant cell an exogenous nucleic acid, that comprises a regulatory regionoperably linked to a nucleotide sequence encoding a polypeptide. The HMMbit score of the amino acid sequence of the polypeptide is greater thanabout 130, 180, 650, 315, or 790 using an HMM generated from the aminoacid sequences depicted in any one of FIG. 1, 2, 3, 4 or 5,respectively. A plant and/or plant tissue produced from the plant cellhas a difference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise the exogenous nucleic acid.

In certain embodiments, the amino acid sequence of the polypeptide hasan HMM score greater than about 180, using an HMM generated from theamino acid sequences depicted in FIG. 2, wherein the polypeptidecomprises an CCT motif domain having 80 percent or greater sequenceidentity to amino acid residues 285 to 329 of SEQ ID NO: 20, residues291 to 335 of SEQ ID NO: 22, residues 235 to 279 of SEQ ID NO: 24,residues 217 to 261 of SEQ ID NO: 26, residues 311 to 355 of SEQ ID NO:28, residues 285 to 329 of SEQ ID NO: 29, residues 281 to 325 of SEQ IDNO: 30, residues 302 to 346 of SEQ ID NO: 32, residues 289 to 333 of SEQID NO: 34, residues 295 to 339 of SEQ ID NO: 36, residues 261 to 305 ofSEQ ID NO: 38, residues 284 to 328 of SEQ ID NO: 40, residues 288 to 332of SEQ ID NO: 42, residues 261 to 305 of SEQ ID NO: 43, residues 239 to283 of SEQ ID NO: 44, residues 294 to 338 of SEQ ID NO: 45, residues 279to 323 of SEQ ID NO: 46, residues 261 to 305 of SEQ ID NO: 47, residues239 to 283 of SEQ ID NO: 48, residues 294 to 338 of SEQ ID NO: 49,residues 261 to 305 of SEQ ID NO: 50, residues 298 to 342 of SEQ ID NO:51, residues 241 to 285 of SEQ ID NO: 52, residues 268 to 312 of SEQ IDNO: 53, residues 245 to 289 of SEQ ID NO: 54, residues 238 to 282 of SEQID NO: 56, residues 245 to 289 of SEQ ID NO: 58, residues 279 to 323 ofSEQ ID NO: 59, residues 236 to 280 of SEQ ID NO: 60, residues 250 to 294of SEQ ID NO: 61, residues 322 to 366 of SEQ ID NO: 62, residues 297 to341 of SEQ ID NO: 63, residues 348 to 392 of SEQ ID NO: 64, residues 312to 356 of SEQ ID NO: 65, residues 340 to 384 of SEQ ID NO: 68, residues307 to 351 of SEQ ID NO: 69, or residues 253 to 297 of SEQ ID NO: 71, orCCT motifs identified in the sequence listing.

In certain embodiments, the amino acid sequence of the polypeptide hasan HMM score greater than about 180, using an HMM generated from theamino acid sequences depicted in FIG. 2, wherein the polypeptidecomprises a B-box zinc finger domain having 80 percent or greatersequence identity to amino acid residues 56 to 103 of SEQ ID NO: 20,residues 62 to 109 of SEQ ID NO: 22, residues 64 to 106 of SEQ ID NO:24, residues 34 to 81 of SEQ ID NO: 26, residues 63 to 110 of SEQ ID NO:28, residues 56 to 103 of SEQ ID NO: 29, residues 56 to 103 of SEQ IDNO: 30, residues 60 to 107 of SEQ ID NO: 32, residues 56 to 103 of SEQID NO: 34, residues 51 to 98 of SEQ ID NO: 36, residues 70 to 112 of SEQID NO: 38, residues 51 to 98 of SEQ ID NO: 40, residues 52 to 99 of SEQID NO: 42, residues 72 to 114 of SEQ ID NO: 43, residues 62 to 104 ofSEQ ID NO: 44, residues 50 to 97 of SEQ ID NO: 45, residues 55 to 102 ofSEQ ID NO: 46, residues 72 to 114 of SEQ ID NO: 47, residues 62 to 104of SEQ ID NO: 48, residues 50 to 97 of SEQ ID NO: 49, residues 27 to 71of SEQ ID NO: 50, residues 60 to 107 of SEQ ID NO: 51, residues 1 to 48of SEQ ID NO: 52, residues 1 to 48 of SEQ ID NO: 53, residues 1 to 48 ofSEQ ID NO: 54, residues 62 to 104 of SEQ ID NO: 56, residues 64 to 106of SEQ ID NO: 58, residues 1 to 48 of SEQ ID NO: 59, residues 61 to 103of SEQ ID NO: 60, residues 70 to 112 of SEQ ID NO: 61, residues 52 to 99of SEQ ID NO: 62, residues 51 to 98 of SEQ ID NO: 63, residues 77 to 119of SEQ ID NO: 64, residues 59 to 106 of SEQ ID NO: 65, residues 59 to106 of SEQ ID NO: 68, residues 34 to 66 of SEQ ID NO: 69, or residues 64to 106 of SEQ ID NO: 71, or a B-box zinc finger domain identified in thesequence listing.

In certain embodiments, the amino acid sequence of the polypeptide hasan HMM score greater than about 650, using an HMM generated from theamino acid sequences depicted in FIG. 3, wherein the polypeptidecomprises an short-chain dehydrogenase domain having 80 percent orgreater sequence identity to amino acid residues 38 to 173 of SEQ ID NO:74, residues 37 to 174 of SEQ ID NO: 76, residues 23 to 160 of SEQ IDNO: 77, residues 7 to 168 of SEQ ID NO: 79, residues 43 to 179 of SEQ IDNO: 81, residues 49 to 188 of SEQ ID NO: 82, residues 48 to 187 of SEQID NO: 83, residues 37 to 172 of SEQ ID NO: 85, residues 35 to 170 ofSEQ ID NO: 86, residues 20 to 160 of SEQ ID NO: 88, or residues 37 to174 of SEQ ID NO: 90, or a short-chain dehydrogenase domain identifiedin the sequence listing.

In certain embodiments, the amino acid sequence of the polypeptide hasan HMM score greater than about 790, using an HMM generated from theamino acid sequences depicted in FIG. 5, wherein the polypeptidecomprises a B3 DNA binding domain and an auxin response factor.

In another aspect, a method comprises introducing into a plant cell anexogenous nucleic acid that comprises a regulatory region operablylinked to a nucleotide sequence encoding a polypeptide having 80 percentor greater sequence identity to an amino acid sequence set forth in SEQID NO: 2, 20, 74, 93 or 112. A plant and/or plant tissue produced fromthe plant cell has a difference in the level of cold tolerance ascompared to the corresponding level of cold tolerance of a control plantthat does not comprise the exogenous nucleic acid.

In another aspect, a method comprises introducing into a plant cell anexogenous nucleic acid, that comprises a regulatory region operablylinked to a nucleotide sequence having 80 percent or greater sequenceidentity to a nucleotide sequence set forth in SEQ ID NO: 1, 19, 92, 97,or 111, or a fragment thereof. A plant and/or plant tissue produced fromthe plant cell has a difference in the level of cold tolerance ascompared to the corresponding level of cold tolerance of a control plantthat does not comprise the exogenous nucleic acid.

Methods of modulating the level of cold tolerance in a plant areprovided herein. In one aspect, a method comprises introducing into aplant cell an exogenous nucleic acid, that comprises a regulatory regionoperably linked to a nucleotide sequence encoding a trans-activatingsmall-interfering RNA (tasiRNA) that acts upon, e.g. suppressesexpression of, an auxin responsive factor (ARF) polypeptide. The HMM bitscore of the amino acid sequence of the ARF polypeptide is greater thanabout 790, using an HMM generated from the amino acid sequences depictedin FIG. 5. A plant and/or plant tissue produced from the plant cell hasa difference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise the exogenous nucleic acid.

Plant cells comprising an exogenous nucleic acid are provided herein. Inone aspect, the exogenous nucleic acid comprises a regulatory regionoperably linked to a nucleotide sequence encoding a tasiRNA. In someembodiments, the nucleotide sequence comprises a tasiRNA coding regionhaving 80 percent or greater sequence identity to a nucleic acidsequence selected from the group consisting of residues 305 to about 346of SEQ ID NO: 111, residues 21 to about 62 of SEQ ID NO: 66, residues 20to about 61 of SEQ ID NO: 67, residues 21 to about 62 of SEQ ID NO: 72,residues 21 to about 62 of SEQ ID NO: 73, residues 77 to about 118 ofSEQ ID NO: 144, residues 292 to about 313 of SEQ ID NO: 145, residues 37to about 78 of SEQ ID NO: 146, residues 56 to about 97 of SEQ ID NO:147, residues 37 to about 78 of SEQ ID NO: 148, residues 45 to about 86of SEQ ID NO: 149, residues 46 to about 98 of SEQ ID NO: 150, residues476 to about 497 of SEQ ID NO: 151, residues 21 to about 62 of SEQ IDNO: 152, residues 21 to about 62 of SEQ ID NO: 153, residues 21 to about62 of SEQ ID NO: 154, residues 21 to about 62 of SEQ ID NO: 155, andresidues 21 to about 62 of SEQ ID NO: 156, wherein a plant produced fromsaid plant cell has a difference in the level of cold tolerance ascompared to the corresponding level of cold tolerance of a control plantthat does not comprise said nucleic acid. Transgenic plants comprisingsuch plant cells are provided herein. In some embodiments, thetransgenic plant comprises an exogenous nucleic acid having a sequenceselected from the group consisting of SEQ ID NO: 66, 67, 72, 73, 111,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, and 156.

Methods of producing a plant and/or plant tissue are provided herein. Inone aspect, a method comprises introducing into a plant cell anexogenous nucleic acid, said exogenous nucleic acid comprising aregulatory region operably linked to a nucleotide sequence encoding agene suppressing tasiRNA, said nucleotide sequence comprising a tasiRNAcoding region having 80 percent or greater sequence identity to anucleic acid sequence selected from the group consisting of residues 305to about 346 of SEQ ID NO: 111, residues 21 to about 62 of SEQ ID NO:66, residues 20 to about 61 of SEQ ID NO: 67, residues 21 to about 62 ofSEQ ID NO: 72, residues 21 to about 62 of SEQ ID NO: 73, residues 77 toabout 118 of SEQ ID NO: 144, residues 292 to about 313 of SEQ ID NO:145, residues 37 to about 78 of SEQ ID NO: 146, residues 56 to about 97of SEQ ID NO: 147, residues 37 to about 78 of SEQ ID NO: 148, residues45 to about 86 of SEQ ID NO: 149, residues 46 to about 98 of SEQ ID NO:150, residues 476 to about 497 of SEQ ID NO: 151, residues 21 to about62 of SEQ ID NO: 152, residues 21 to about 62 of SEQ ID NO: 153,residues 21 to about 62 of SEQ ID NO: 154, residues 21 to about 62 ofSEQ ID NO: 155, and residues 21 to about 62 of SEQ ID NO: 156, wherein aplant produced from said plant cell has a difference in the level ofcold tolerance as compared to the corresponding level of cold toleranceof a control plant that does not comprise said nucleic acid. In certainembodiments, the expression of a target ARF gene is suppressed in aplant. In some embodiments, the ARF gene encodes a polypeptide having 80percent or greater sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 112, 114, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 134, 136,137, 138, 139, 141, and 143. In other embodiments, the ARF gene encodesa polypeptide and the HMM bit score of the amino acid sequence of saidpolypeptide is greater than about 790, said HMM based on the amino acidsequences depicted in FIG. 5. In other embodiments, the gene suppressingtasiRNA or its complement is complementary to RNA transcribed from saidtarget ARF gene. In other embodiments, the nucleotide sequence encodinga gene suppressing tasiRNA comprises a microRNA recognition site having80 percent or greater sequence identity to a nucleic acid sequenceselected from the group consisting of residues 109 to about 129 of SEQID NO: 66, residues 114 to about 135 of SEQ ID NO: 67, residues 119 toabout 139 of SEQ ID NO: 72, residues 108 to about 128 of SEQ ID NO: 73,residues 234 to about 254 of SEQ ID NO: 144, residues 135 to about 176of SEQ ID NO: 145, residues 173 to about 189 of SEQ ID NO: 147, residues154 to about 170 of SEQ ID NO: 148, residues 134 to about 157 of SEQ IDNO: 149, residues 154 to about 198 of SEQ ID NO: 150, residues 319 toabout 360 of SEQ ID NO: 151, residues 121 to about 141 of SEQ ID NO:152, residues 120 to about 140 of SEQ ID NO: 153, residues 121 to about141 of SEQ ID NO: 154, residues 121 to about 141 of SEQ ID NO: 155,residues 121 to about 141 of SEQ ID NO: 156, and residues 462 to about483 of SEQ ID NO: 111.

Plant cells comprising an exogenous nucleic acid are provided herein. Inone aspect, the exogenous nucleic acid comprises a regulatory regionoperably linked to a nucleotide sequence encoding a polypeptide. The HMMbit score of the amino acid sequence of the polypeptide is greater thanabout 130, using an HMM based on the amino acid sequences depicted inone of FIG. 1, 2, 3, 4, or 5. The plant and/or plant cells has adifference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise the exogenous nucleic acid. In another aspect, the exogenousnucleic acid comprises a regulatory region operably linked to anucleotide sequence encoding a polypeptide having 80 percent or greatersequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 20, 93, or 74. A plant and/or plant tissueproduced from the plant cell has a difference in the level of coldtolerance as compared to the corresponding level of cold tolerance of acontrol plant that does not comprise the exogenous nucleic acid. Inanother aspect, the exogenous nucleic acid comprises a regulatory regionoperably linked to a nucleotide sequence having 80 percent or greatersequence identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, 19, 92, 97, or 111. A plant and/or planttissue produced from the plant cell has a difference in the level ofcold tolerance as compared to the corresponding level of cold toleranceof a control plant that does not comprise the exogenous nucleic acid. Atransgenic plant comprising such a plant cell is also provided. Alsoprovided is a seed product. The product comprises embryonic tissue froma transgenic plant.

Isolated nucleic acids are also provided. In one aspect, an isolatednucleic acid comprises a nucleotide sequence having 80% or greatersequence identity to the nucleotide sequence set forth in SEQ ID NO: 3,5, 7, 9, 11, 14, 16, 21, 23, 25, 27, 33, 35, 37, 39, 41, 55, 57, 70, 75,80, 84, 87, 91, 92, 97, 105, 113, 115, 129, 133, 140, or 142.

In another aspect, an isolated nucleic acid comprises a nucleotidesequence encoding a polypeptide having 80% or greater sequence identityto the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 22,24, 26, 28, 36, 38, 40, 42, 71, 74, 85, 88, 93, 105, 114, 116, 130, 134,136, 141, or 143.

In another aspect, methods of identifying a genetic polymorphismassociated with variation in the level of cold tolerance are provided.The methods include providing a population of plants, and determiningwhether one or more genetic polymorphisms in the population aregenetically linked to the locus for a polypeptide selected from thegroup consisting of the polypeptides depicted in FIGS. 1, 2, 3, 4, and 5and functional homologs thereof, such as, but not limited to, thoseidentified in the Sequence Listing. The correlation between variation inthe level of cold tolerance in a tissue in plants of the population andthe presence of the one or more genetic polymorphisms in plants of thepopulation is measured, thereby permitting identification of whether ornot the one or more genetic polymorphisms are associated with suchvariation.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of Ceres SEEDLINE ID no.ME00327 with homologousand/or orthologous amino acid sequences Ceres SEEDLINE ID no.ME00327(SEQ ID NO: 2), Ceres CLONE ID no.1915941 (SEQ ID NO: 8), Ceres ANNOT IDno.1461830 (SEQ ID NO: 10), Ceres CLONE ID no.1080942 (SEQ ID NO: 15),and Ceres CLONE ID no.1073190 (SEQ ID NO: 17). In all the alignmentfigures shown herein, a dash in an aligned sequence represents a gap,i.e., a lack of an amino acid at that position. Identical amino acids orconserved amino acid substitutions among aligned sequences areidentified by boxes. FIG. 1 and the other alignment figures providedherein were generated using the program MUSCLE version 3.52.

FIG. 2 is an alignment of Ceres SEEDLINE ID no.ME04315 (SEQ ID NO: 20)with homologous and/or orthologous amino acid sequences Ceres CLONE IDno.1842825 (SEQ ID NO: 22), Ceres ANNOT ID no.1482536 (SEQ ID NO: 28),Ceres CLONE ID no.463157 (SEQ ID NO: 32), Ceres CLONE ID no.1674443 (SEQID NO:40), GI ID no.116310719 (SEQ ID NO: 44), Ceres CLONE ID no.907473(SEQ ID NO: 58), and Ceres CLONE ID no.1755065 (SEQ ID NO: 71).

FIG. 3 is an alignment of full length homologous and/or orthologousamino acid sequences of Ceres SEEDLINE ID no.ME17294 (SEQ ID NO: 93),including Ceres CLONE ID no.473040 (SEQ ID NO: 79), Ceres CLONE IDno.922223 (SEQ ID NO: 81), GI ID no.125528967 (SEQ ID NO: 82), GI IDno.125573200 (SEQ ID NO: 83), Ceres ANNOT ID no.1527409 (SEQ ID NO: 85),GI ID no.92871098 (SEQ ID NO: 86), Ceres CLONE ID no.1831117 (SEQ ID NO:88), and Ceres ANNOT ID no.857222 (SEQ ID NO: 90).

FIG. 4 is an alignment of a truncated version of Ceres SEEDLINE IDno.ME17294 (SEQ ID NO: 93) with homologous and/or orthologous amino acidtruncated sequences, Ceres CLONE ID no.1831117 (SEQ ID NO: 95), CeresCLONE ID no.1844076 (SEQ ID NO: 98), Ceres CLONE ID no.473040 (SEQ IDNO:104), Ceres CLONE ID no.922223 (SEQ ID NO: 106), and GI IDno.125528967 (SEQ ID NO:107).

FIG. 5 is an alignment of functional homologs of the ARF (Auxin ResponseFactor) genes ARF2, ARF3, and ARF4, including LOCUS ID no. AT5G62000(SEQ ID NO: 112), Ceres ANNOT ID no.1527370 (SEQ ID NO: 114), CeresANNOT ID no.1473961 (SEQ ID NO: 116), GI ID no.62319853 (SEQ ID NO:117), GI ID no.62319903 (SEQ ID NO:118), GI ID no.47716275 (SEQ ID NO:119), GI ID no.125534572 (SEQ ID NO:120), GI ID no.26251300(SEQ IDNO:121), GI ID no.115441981 (SEQ ID NO:123), GI ID no.23893346 (SEQ IDNO:124), GI ID no.115485689 (SEQ ID NO:125), GI ID no.108864435 (SEQ IDNO:126), GI ID no.50511471 (SEQ ID NO:127), LOCUS ID no. At2g33860 (SEQID NO:128), GI ID no.2245390 (SEQ ID NO:131), GI ID no.3228517 (SEQ IDNO:132), Ceres CLONE ID no.827306 (SEQ ID NO: 134), Ceres CLONE IDno.1598488 (SEQ ID NO: 136), GI ID no.125553314 (SEQ ID NO:138), andCeres CLONE ID no.462443 (SEQ ID NO:143).

DETAILED DESCRIPTION

The invention features methods and materials related to modulating coldtolerance levels in plants. In some embodiments, the cold toleranceplants of the invention, under cold stress and/or cold flux conditions,have modulated levels of growth, cold acclimation, and/or cold damage.The methods can include transforming a plant cell with a nucleic acidencoding a cold tolerance modulating polypeptide, wherein expression ofthe polypeptide results in a modulated level of cold tolerance. Plantcells produced using such methods can be grown to produce plants havingan increased cold tolerance. Such plants, and the seeds of such plants,may be used to produce, for example, plants and/or plant tissues havingincreased biomass.

I. Definitions

“Amino acid” refers to one of the twenty biologically occurring aminoacids and to synthetic amino acids, including D/L optical isomers.

“Cell type-preferential promoter” or “tissue-preferential promoter”refers to a promoter that drives expression preferentially in a targetcell type or tissue, respectively, but may also lead to sometranscription in other cell types or tissues as well.

“Cold.” Plant species vary in their capacity to tolerate lowtemperatures. Chilling-sensitive plant species, including manyagronomically important species, can be injured by cold, above-freezingtemperatures. At temperatures below the freezing-point of water mostplant species will be damaged. Thus, “cold” can be defined as thetemperature at which a given plant species will be adversely affected asevidenced by symptoms such as decreased photosynthesis and membranedamage (measured by electrolyte leakage). Since plant species vary intheir capacity to tolerate cold, the precise environmental conditionsthat cause cold stress cannot be generalized. However, cold tolerantplants are characterized by their ability to retain their normalappearance, recover quickly from low temperature conditions, exhibitnormal or increased growth under low temperature conditions, and/or haveimproved cold acclimation. Such cold tolerant plants produce higherbiomass and/or yield than plants that are not cold tolerant. Differencesin physical appearance, recovery and yield can be quantified andstatistically analyzed using well known measurement and analysismethods.

“Control plant” refers to a plant that does not contain the exogenousnucleic acid present in a transgenic plant of interest, but otherwisehas the same or similar genetic background as such a transgenic plant. Asuitable control plant can be a non-transgenic wild type plant, anon-transgenic segregant from a transformation experiment, or atransgenic plant that contains an exogenous nucleic acid other than theexogenous nucleic acid of interest.

“Domains” are groups of substantially contiguous amino acids in apolypeptide that can be used to characterize protein families and/orparts of proteins. Such domains have a “fingerprint” or “signature” thatcan comprise conserved primary sequence, secondary structure, and/orthree-dimensional conformation. Generally, domains are correlated withspecific in vitro and/or in vivo activities. A domain can have a lengthof from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids,or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 aminoacids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400amino acids.

“Down-regulation” refers to regulation that decreases production ofexpression products (mRNA, polypeptide, or both) relative to basal ornative states.

“Exogenous” with respect to a nucleic acid indicates that the nucleicacid is part of a recombinant nucleic acid construct, or is not in itsnatural environment. For example, an exogenous nucleic acid can be asequence from one species introduced into another species, i.e., aheterologous nucleic acid. Typically, such an exogenous nucleic acid isintroduced into the other species via a recombinant nucleic acidconstruct. An exogenous nucleic acid can also be a sequence that isnative to an organism and that has been reintroduced into cells of thatorganism. An exogenous nucleic acid that includes a native sequence canoften be distinguished from the naturally occurring sequence by thepresence of non-natural sequences linked to the exogenous nucleic acid,e.g., non-native regulatory sequences flanking a native sequence in arecombinant nucleic acid construct. In addition, stably transformedexogenous nucleic acids typically are integrated at positions other thanthe position where the native sequence is found. It will be appreciatedthat an exogenous nucleic acid may have been introduced into aprogenitor and not into the cell under consideration. For example, atransgenic plant containing an exogenous nucleic acid can be the progenyof a cross between a stably transformed plant and a non-transgenicplant. Such progeny are considered to contain the exogenous nucleicacid.

“Expression” refers to the process of converting genetic information ofa polynucleotide into RNA through transcription, which is catalyzed byan enzyme, RNA polymerase, and into protein, through translation of mRNAon ribosomes.

“Heterologous polypeptide” as used herein refers to a polypeptide thatis not a naturally occurring polypeptide in a plant cell, e.g., atransgenic Panicum virgatum plant transformed with and expressing thecoding sequence for a nitrogen transporter polypeptide from a Zea maysplant.

“Isolated nucleic acid” as used herein includes a naturally-occurringnucleic acid, provided one or both of the sequences immediately flankingthat nucleic acid in its naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, anucleic acid that exists as a purified molecule or a nucleic acidmolecule that is incorporated into a vector or a virus. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries, genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

“Modulation” of the level of a cold tolerance refers to the change inthe level of the indicated compound or constituent that is observed as aresult of expression of, or transcription from, an exogenous nucleicacid in a plant cell. The change in level is measured relative to thecorresponding level in control plants.

“Nucleic acid” and “polynucleotide” are used interchangeably herein, andrefer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA,and DNA or RNA containing nucleic acid analogs. Polynucleotides can haveany three-dimensional structure. A nucleic acid can be double-strandedor single-stranded (i.e., a sense strand or an antisense strand).Non-limiting examples of polynucleotides include genes, gene fragments,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, nucleic acid probes and nucleic acid primers. Apolynucleotide may contain unconventional or modified nucleotides.

“Operably linked” refers to the positioning of a regulatory region and asequence to be transcribed in a nucleic acid so that the regulatoryregion is effective for regulating transcription or translation of thesequence. For example, to operably link a coding sequence and aregulatory region, the translation initiation site of the translationalreading frame of the coding sequence is typically positioned between oneand about fifty nucleotides downstream of the regulatory region. Aregulatory region can, however, be positioned as much as about 5,000nucleotides upstream of the translation initiation site, or about 2,000nucleotides upstream of the transcription start site.

“Polypeptide” as used herein refers to a compound of two or more subunitamino acids, amino acid analogs, or other peptidomimetics, regardless ofpost-translational modification, e.g., phosphorylation or glycosylation.The subunits may be linked by peptide bonds or other bonds such as, forexample, ester or ether bonds. Full-length polypeptides, truncatedpolypeptides, point mutants, insertion mutants, splice variants,chimeric proteins, and fragments thereof are encompassed by thisdefinition.

“Progeny” includes descendants of a particular plant or plant line.Progeny of an instant plant include seeds formed on F₁, F₂, F₃, F₄, F₅,F₆ and subsequent generation plants, or seeds formed on BC₁, BC₂, BC₃,and subsequent generation plants, or seeds formed on F₁BC₁, F₁BC₂,F₁BC₃, and subsequent generation plants. The designation F₁ refers tothe progeny of a cross between two parents that are geneticallydistinct. The designations F₂, F₃, F₄, F₅ and F₆ refer to subsequentgenerations of self- or sib-pollinated progeny of an F₁ plant.

“Regulatory region” refers to a nucleic acid having nucleotide sequencesthat influence transcription or translation initiation and rate, andstability and/or mobility of a transcription or translation product.Regulatory regions include, without limitation, promoter sequences,enhancer sequences, response elements, protein recognition sites,inducible elements, protein binding sequences, 5′ and 3′ untranslatedregions (UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, introns, and combinations thereof. Aregulatory region typically comprises at least a core (basal) promoter.A regulatory region also may include at least one control element, suchas an enhancer sequence, an upstream element or an upstream activationregion (UAR). For example, a suitable enhancer is a cis-regulatoryelement (-212 to -154) from the upstream region of the octopine synthase(ocs) gene. Fromm et al., The Plant Cell, 1:977-984 (1989).

“Up-regulation” refers to regulation that increases the level of anexpression product (mRNA, polypeptide, or both) relative to basal ornative states.

“Vector” refers to a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. The term“vector” includes cloning and expression vectors, as well as viralvectors and integrating vectors. An “expression vector” is a vector thatincludes a regulatory region.

II. Polypeptides

Polypeptides described herein include cold tolerance-modulatingpolypeptides. Cold tolerance-modulating polypeptides can be effective tomodulate cold tolerance levels when expressed in a plant or plant cell.Such polypeptides typically contain at least one domain indicative ofcold tolerance-modulating polypeptides, as described in more detailherein. Cold tolerance-modulating polypeptides typically have an HMM bitscore that is greater than 130, as described in more detail herein. Insome embodiments, cold tolerance-modulating polypeptides have greaterthan 80% identity to SEQ ID NOs: 2, 20, 74, 93 or 112, as described inmore detail herein.

A. Domains Indicative of Cold Tolerance-Modulating Polypeptides

A cold tolerance-modulating polypeptide can contain a B-box zinc fingerdomain. The B-box zinc finger domain is often found associated with CCTmotif. SEQ ID NO: 20 sets forth the amino acid sequence of anArabidopsis clone, identified herein as Ceres SEEDLINE ID no.ME04315(SEQ ID NO: 20), that is predicted to encode a polypeptide containing aCCT motif and a B-box zinc finger domain. A B-box zinc finger domain istypically around 40 amino acids in length. This motif is generallyassociated with a finger. It is found, for example, in transcriptionfactors, ribonucleoproteins and protooncoproteins. It has been shown tobe essential but not sufficient to localize the PML protein in apunctate pattern in interphase nuclei. Among the 7 possible ligands forthe zinc atom contained in a B-box, only 4 are used and bind one zincatom in a Cys2-His2 tetrahedral arrangement. The NMR analysis revealsthat the B-box structure comprises two beta-strands, two helical turnsand three extended loop regions different from any other zinc bindingmotif. A CCT motif can be found in a number of plant proteins. It isrich in basic amino acids and has been called a CCT motif after Co, Co1and Toc1. The CCT motif is about 45 amino acids long and contains aputative nuclear localization signal within the second half of the CCTmotif. Toc1 mutants have been identified in this region. The CCT(CONSTANS, CO-like, and TOC1) domain is a highly conserved basic moduleof ˜43 amino acids, which is found near the C-terminus of plantproteins. The CCT domain is often found in association with otherdomains, such as the B-box zinc finger, the GATA-type zinc finger, theZIM motif or the response regulatory domain. The CCT domain contains aputative nuclear localization signal within the second half of the CCTmotif and has been shown to be involved in nuclear localization andprobably also has a role in protein-protein interaction.

In embodiments of the invention, a cold tolerance-modulating polypeptidecan comprise a CCT motif having 80% or greater sequence identity toamino acid residues 285 to 329 of SEQ ID NO: 20, residues 291 to 335 ofSEQ ID NO: 22, residues 235 to 279 of SEQ ID NO: 24, residues 217 to 261of SEQ ID NO: 26, residues 311 to 355 of SEQ ID NO: 28, residues 285 to329 of SEQ ID NO: 29, residues 281 to 325 of SEQ ID NO: 30, residues 302to 346 of SEQ ID NO: 32, residues 289 to 333 of SEQ ID NO: 34, residues295 to 339 of SEQ ID NO: 36, residues 261 to 305 of SEQ ID NO: 38,residues 284 to 328 of SEQ ID NO: 40, residues 288 to 332 of SEQ ID NO:42, residues 261 to 305 of SEQ ID NO: 43, residues 239 to 283 of SEQ IDNO: 44, residues 294 to 338 of SEQ ID NO: 45, residues 279 to 323 of SEQID NO: 46, residues 261 to 305 of SEQ ID NO: 47, residues 239 to 283 ofSEQ ID NO: 48, residues 294 to 338 of SEQ ID NO: 49, residues 261 to 305of SEQ ID NO: 50, residues 298 to 342 of SEQ ID NO: 51, residues 241 to285 of SEQ ID NO: 52, residues 268 to 312 of SEQ ID NO: 53, residues 245to 289 of SEQ ID NO: 54, residues 238 to 282 of SEQ ID NO: 56, residues245 to 289 of SEQ ID NO: 58, residues 279 to 323 of SEQ ID NO: 59,residues 236 to 280 of SEQ ID NO: 60, residues 250 to 294 of SEQ ID NO:61, residues 322 to 366 of SEQ ID NO: 62, residues 297 to 341 of SEQ IDNO: 63, residues 348 to 392 of SEQ ID NO: 64, residues 312 to 356 of SEQID NO: 65, residues 340 to 384 of SEQ ID NO: 68, residues 307 to 351 ofSEQ ID NO: 69, or residues 253 to 297 of SEQ ID NO: 71, or a CCT motifidentified in the sequence listing.

In embodiments of the invention, a cold tolerance-modulating polypeptidecan comprise a B-box zinc finger domain having 80% or greater sequenceidentity to amino acid residues 56 to 103 of SEQ ID NO: 20, residues 62to 109 of SEQ ID NO: 22, residues 64 to 106 of SEQ ID NO: 24, residues34 to 81 of SEQ ID NO: 26, residues 63 to 110 of SEQ ID NO: 28, residues56 to 103 of SEQ ID NO: 29, residues 56 to 103 of SEQ ID NO: 30,residues 60 to 107 of SEQ ID NO: 32, residues 56 to 103 of SEQ ID NO:34, residues 51 to 98 of SEQ ID NO: 36, residues 70 to 112 of SEQ ID NO:38, residues 51 to 98 of SEQ ID NO: 40, residues 52 to 99 of SEQ ID NO:42, residues 72 to 114 of SEQ ID NO: 43, residues 62 to 104 of SEQ IDNO: 44, residues 50 to 97 of SEQ ID NO: 45, residues 55 to 102 of SEQ IDNO: 46, residues 72 to 114 of SEQ ID NO: 47, residues 62 to 104 of SEQID NO: 48, residues 50 to 97 of SEQ ID NO: 49, residues 27 to 71 of SEQID NO: 50, residues 60 to 107 of SEQ ID NO: 51, residues 1 to 48 of SEQID NO: 52, residues 1 to 48 of SEQ ID NO: 53, residues 1 to 48 of SEQ IDNO: 54, residues 62 to 104 of SEQ ID NO: 56, residues 64 to 106 of SEQID NO: 58, residues 1 to 48 of SEQ ID NO: 59, residues 61 to 103 of SEQID NO: 60, residues 70 to 112 of SEQ ID NO: 61, residues 52 to 99 of SEQID NO: 62, residues 51 to 98 of SEQ ID NO: 63, residues 77 to 119 of SEQID NO: 64, residues 59 to 106 of SEQ ID NO: 65, residues 59 to 106 ofSEQ ID NO: 68, residues 34 to 66 of SEQ ID NO: 69, or residues 64 to 106of SEQ ID NO: 71, or a B-box zinc finger domain identified in thesequence listing.

A cold tolerance-modulating polypeptide can contain a short-chaindehydrogenase domain The motif is present in SEQ ID NO: 93, which setsforth the amino acid sequence of an Arabidopsis clone, identified hereinas Ceres SEEDLINE ID no.ME17294 (SEQ ID NO: 93), that is predicted toencode a polypeptide containing a short-chain dehydrogenase domain. Theshort-chain dehydrogenases/reductases family (SDR) is a very largefamily of enzymes, most of which are known to be NAD- or NADP-dependentoxidoreductases. As the first member of this family to be characterizedwas Drosophila alcohol dehydrogenase, this family used to be called‘insect-type’, or ‘short-chain’ alcohol dehydrogenases. Most members ofthis family are proteins of about 250 to 300 amino acid residues.

In embodiments of the invention, a cold tolerance-modulating polypeptidecan comprise a short-chain dehydrogenase domain having 80% or greateridentity to amino acid residues 38 to 173 of SEQ ID NO: 74, residues 37to 174 of SEQ ID NO: 76, residues 23 to 160 of SEQ ID NO: 77, residues 7to 168 of SEQ ID NO: 79, residues 43 to 179 of SEQ ID NO: 81, residues49 to 188 of SEQ ID NO: 82, residues 48 to 187 of SEQ ID NO: 83,residues 37 to 172 of SEQ ID NO: 85, residues 35 to 170 of SEQ ID NO:86, residues 20 to 160 of SEQ ID NO: 88, or residues 37 to 174 of SEQ IDNO: 90, or a short-chain dehydrogenase domain identified in the sequencelisting.

In some embodiments, a cold tolerance-modulating polypeptide istruncated at the amino- or carboxy-terminal end of a naturally occurringpolypeptide. A truncated polypeptide may retain certain domains of thenaturally occurring polypeptide while lacking others. Thus, lengthvariants that are up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, or 170 amino acids shorter or longer typicallyexhibit the cold tolerance-modulating activity of a truncatedpolypeptide. In some embodiments, a truncated polypeptide is a dominantnegative polypeptide. SEQ ID NO: 93 sets forth the amino sequence of acold tolerance-modulating polypeptide that is truncated at theamino-terminal end relative to a naturally occurring polypeptide.Expression in a plant and/or plant tissue of such a truncatedpolypeptide confers a difference in the level of cold tolerance in aplant and/or tissue of the plant as compared to the corresponding levelin tissue of a control plant.

B. Functional Homologs Identified by Reciprocal BLAST

In some embodiments, one or more functional homologs of a reference coldtolerance-modulating polypeptide defined by one or more of the Pfamdescriptions indicated above are suitable for use as coldtolerance-modulating polypeptides. A functional homolog is a polypeptidethat has sequence similarity to a reference polypeptide, and thatcarries out one or more of the biochemical or physiological function(s)of the reference polypeptide. A functional homolog and the referencepolypeptide may be natural occurring polypeptides, and the sequencesimilarity may be due to convergent or divergent evolutionary events. Assuch, functional homologs are sometimes designated in the literature ashomologs, or orthologs, or paralogs. Variants of a naturally occurringfunctional homolog, such as polypeptides encoded by mutants of a wildtype coding sequence, may themselves be functional homologs. Functionalhomologs can also be created via site-directed mutagenesis of the codingsequence for a cold tolerance-modulating polypeptide, or by combiningdomains from the coding sequences for different naturally-occurring coldtolerance-modulating polypeptides (“domain swapping”). The term“functional homolog” is sometimes applied to the nucleic acid thatencodes a functionally homologous polypeptide.

Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofcold tolerance-modulating polypeptides. Sequence analysis can involveBLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundant databasesusing a cold tolerance-modulating polypeptide amino acid sequence as thereference sequence. Amino acid sequence is, in some instances, deducedfrom the nucleotide sequence. Those polypeptides in the database thathave greater than 40% sequence identity are candidates for furtherevaluation for suitability as a cold tolerance-modulating polypeptideAmino acid sequence similarity allows for conservative amino acidsubstitutions, such as substitution of one hydrophobic residue foranother or substitution of one polar residue for another. If desired,manual inspection of such candidates can be carried out in order tonarrow the number of candidates to be further evaluated. Manualinspection can be performed by selecting those candidates that appear tohave domains present in cold tolerance-modulating polypeptides, e.g.,conserved functional domains.

Conserved regions can be identified by locating a region within theprimary amino acid sequence of a cold tolerance-modulating polypeptidethat is a repeated sequence, forms some secondary structure (e.g.,helices and beta sheets), establishes positively or negatively chargeddomains, or represents a protein motif or domain. See, e.g., the Pfamweb site describing consensus sequences for a variety of protein motifsand domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ andpfam.janelia.org/. A description of the information included at the Pfamdatabase is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322(1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman etal., Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can bedetermined by aligning sequences of the same or related polypeptidesfrom closely related species. Closely related species preferably arefrom the same family. In some embodiments, alignment of sequences fromtwo different species is adequate.

Typically, polypeptides that exhibit at least about 40% amino acidsequence identity are useful to identify conserved regions. Conservedregions of related polypeptides exhibit at least 45% amino acid sequenceidentity (e.g., at least 50%, at least 60%, at least 70%, at least 80%,or at least 90% amino acid sequence identity). In some embodiments, aconserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acidsequence identity.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 2 are provided in FIG. 1 and in theSequence Listing. Such exemplary functional homologs include Ceres CLONEID no.1897908 (SEQ ID NO:4), Ceres CLONE ID no.1938030 (SEQ ID NO: 6),Ceres CLONE ID no.1915941 (SEQ ID NO: 8), Ceres ANNOT ID no.1461830 (SEQID NO: 10), Ceres ANNOT ID no.1439985 (SEQ ID NO: 12), GI ID no.15241794(SEQ ID NO: 13), Ceres CLONE ID no.1080942 (SEQ ID NO:15), and CeresCLONE ID no.1073190 (SEQ ID NO:17). In some cases, a functional homologof SEQ ID NO: 2 has an amino acid sequence with at least 20% sequenceidentity, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 52%, 56%, 59%, 61%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to theamino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 15,or 17.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 20 are provided in FIG. 2 and in theSequence Listing. Such exemplary functional homologs include Ceres CLONEID no.1842825 (SEQ ID NO: 22), Ceres CLONE ID no.1834027 (SEQ ID NO:24), Ceres CLONE ID no.1837064 (SEQ ID NO: 26), Ceres ANNOT IDno.1482536 (SEQ ID NO: 28), GI ID no.18424009 (SEQ ID NO: 29), GI IDno.9759262 (SEQ ID NO: 30), Ceres CLONE ID no.463157 (SEQ ID NO: 32),Ceres CLONE ID no.685991 (SEQ ID NO: 34), Ceres CLONE ID no.702632 (SEQID NO: 36), Ceres CLONE ID no.1559496 (SEQ ID NO: 38), Ceres CLONE IDno.1674443 (SEQ ID NO: 40), Ceres CLONE ID no.1828897 (SEQ ID NO: 42),GI ID no.125540249 (SEQ ID NO: 43), GI ID no.116310719 (SEQ ID NO: 44),GI ID no.125556324 (SEQ ID NO: 45), GI ID no.125538317 (SEQ ID NO: 46),GI ID no.115447239 (SEQ ID NO: 47), GI ID no.115459216 (SEQ ID NO: 48),GI ID no.115469296 (SEQ ID NO: 49), GI ID no.125582846 (SEQ ID NO: 50),GI ID no.92875402 (SEQ ID NO: 51), GI ID no.3341723 (SEQ ID NO: 52), GIID no.4091806 (SEQ ID NO: 53), GI ID no.60459257 (SEQ ID NO: 54), CeresCLONE ID no.1756710 (SEQ ID NO: 56), Ceres CLONE ID no.907473 (SEQ IDNO: 58), GI ID no.4091804 (SEQ ID NO: 59), GI ID no.21667487 (SEQ ID NO:60), GI ID no.21655154 (SEQ ID NO: 61), GI ID no.45544883 (SEQ ID NO:62), GI ID no.21655166 (SEQ ID NO: 63), GI ID no.10946337 (SEQ ID NO:64), GI ID no.90657642 (SEQ ID NO: 65), GI ID no.45544887 (SEQ ID NO:68), GI ID no.47606678 (SEQ ID NO: 69), or Ceres CLONE ID no.1755065(SEQ ID NO: 71). In some cases, a functional homolog of SEQ ID NO: 20has an amino acid sequence with at least 20% sequence identity, e.g.,25%, 30%, 35%, 40%, 45%, 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acidsequence set forth in SEQ ID NO: 20, 22, 24, 26, 28, 29, 30, 32, 34, 36,38, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 58, 59,60, 61, 62, 63, 64, 65, 68, 69, or 71.

Examples of amino acid sequences of full length functional homologs ofthe truncated polypeptide set forth in SEQ ID NO: 93 are provided inFIG. 3 and in the Sequence Listing. Such exemplary functional homologsinclude Ceres CLONE ID no.1844076 (SEQ ID NO: 74), Ceres CLONE IDno.35974 (SEQ ID NO: 76), GI ID no.10176876 (SEQ ID NO: 77), Ceres CLONEID no.473040 (SEQ ID NO: 79), Ceres CLONE ID no.922223 (SEQ ID NO: 81),GI ID no.125528967 (SEQ ID NO: 82), GI ID no.125573200 (SEQ ID NO: 83),Ceres ANNOT ID no.1527409 (SEQ ID NO: 85), GI ID no.92871098 (SEQ ID NO:86), Ceres CLONE ID no.1831117 (SEQ ID NO: 88), and Ceres ANNOT IDno.857222 (SEQ ID NO: 90). In some cases, a full length functionalhomolog of SEQ ID NO: 93 has an amino acid sequence with at least 20%sequence identity, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 74, 76, 77,79, 81, 82, 83, 85, 86, 88, 90, or 93.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 93 are provided in FIG. 4 and in theSequence Listing. Such exemplary functional homologs include Ceres ANNOTID no.857222 (SEQ ID NO: 110), Ceres CLONE ID no.1831117 (SEQ ID NO:95), GI ID no.92871098 (SEQ ID NO: 96), Ceres CLONE ID no.1844076 (SEQID NO: 98), Ceres CLONE ID no.35974 (SEQ ID NO: 100), GI ID no.110737329(SEQ ID NO: 101), GI ID no.10176876 (SEQ ID NO: 102), Ceres CLONE IDno.473040 (SEQ ID NO: 104), Ceres CLONE ID no.922223 (SEQ ID NO: 106),GI ID no.125528967 (SEQ ID NO: 107), and GI ID no.115442007 (SEQ ID NO:108). In some cases, a functional homolog of SEQ ID NO: 93 has an aminoacid sequence with at least 20% sequence identity, e.g., 25%, 30%, 35%,40%, 45%, 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, or 99% sequence identity, to the amino acid sequence set forthin SEQ ID NO: 93, 110, 95, 96, 98, 100, 101, 102, 104, 106, 107, or 108.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 116 are provided in FIG. 5 and inthe Sequence Listing. Such exemplary functional homologs include LOCUSID no. AT5G62000 (SEQ ID NO: 112), Ceres ANNOT ID no.1527370 (SEQ ID NO:114), GI ID no.62319853 (SEQ ID NO: 117), GI ID no.62319903 (SEQ ID NO:118), GI ID no.47716275 (SEQ ID NO: 119), GI ID no.125534572 (SEQ ID NO:120), GI ID no.26251300 (SEQ ID NO: 121), GI ID no.125528952 (SEQ ID NO:122), GI ID no.115441981 (SEQ ID NO: 123), GI ID no.23893346 (SEQ ID NO:124), GI ID no.115485689 (SEQ ID NO: 125), GI ID no.108864435 (SEQ IDNO: 126), GI ID no.50511471 (SEQ ID NO: 127), LOCUS ID no. At2g33860(SEQ ID NO: 128), Ceres ANNOT ID no.1536494 (SEQ ID NO: 130), GI IDno.2245390 (SEQ ID NO: 131), GI ID no.3228517 (SEQ ID NO: 132), CeresCLONE ID no.827306 (SEQ ID NO: 134), Ceres CLONE ID no.1598488 (SEQ IDNO: 136), GI ID no.125527740 (SEQ ID NO: 137), GI ID no.125553314 (SEQID NO: 138), LOCUS ID no. At5g60450 (SEQ ID NO: 139), Ceres ANNOT IDno.1515383 (SEQ ID NO: 141), and Ceres CLONE ID no.462443 (SEQ ID NO:143). In some cases, a functional homolog of SEQ ID NO: 116 has an aminoacid sequence with at least 20% sequence identity, e.g., 25%, 30%, 35%,40%, 45%, 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, or 99% sequence identity, to the amino acid sequence set forthin SEQ ID NO: 116, 112, 114, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 130, 131, 132, 134, 136, 137, 138, 139, 141, or 143.

Examples of nucleic acid sequences of functional homologs of the tasiRNAencoding nucleic acid sequence set forth in SEQ ID NO: 111 are found inthe Sequence Listing. Such exemplary functional homologs include 66, 67,72, 73, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, and156. In some cases, a functional homolog of SEQ ID NO: 111 has annucleic acid sequence with at least 20% sequence identity, e.g., 25%,30%, 35%, 40%, 45%, 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleic acidsequence set forth in SEQ ID NO: 111, 66, 67, 72, 73, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, or 156.

The identification of conserved regions in a cold tolerance-modulatingpolypeptide facilitates production of variants of coldtolerance-modulating polypeptides. Variants of cold tolerance-modulatingpolypeptides typically have 10 or fewer conservative amino acidsubstitutions within the primary amino acid sequence, e.g., 7 or fewerconservative amino acid substitutions, 5 or fewer conservative aminoacid substitutions, or between 1 and 5 conservative substitutions. Auseful variant polypeptide can be constructed based on one of thealignments set forth in FIG. 1, FIG. 2, FIG. 3, FIG. 4, or FIG. 5,and/or homologs identified in the Sequence Listing. Such a polypeptideincludes the conserved regions, arranged in the order depicted in theFigures from amino-terminal end to carboxy-terminal end. Such apolypeptide may also include zero, one, or more than one amino acid inpositions marked by dashes. When no amino acids are present at positionsmarked by dashes, the length of such a polypeptide is the sum of theamino acid residues in all conserved regions. When amino acids arepresent at all positions marked by dashes, such a polypeptide has alength that is the sum of the amino acid residues in all conservedregions and all dashes.

C. Functional Homologs Identified by HMMER

In some embodiments, useful cold tolerance-modulating polypeptidesinclude those that fit a Hidden Markov Model based on the polypeptidesset forth in any one of FIGS. 1-4 or ARFs that are acted upon by tasiRNA(FIG. 5). A Hidden Markov Model (HMM) is a statistical model of aconsensus sequence for a group of functional homologs. See, Durbin etal., Biological Sequence Analysis: Probabilistic Models of Proteins andNucleic Acids, Cambridge University Press, Cambridge, UK (1998). An HMMis generated by the program HMMER 2.3.2 with default program parameters,using the sequences of the group of functional homologs as input. Themultiple sequence alignment is generated by ProbCons (Do et al., GenomeRes., 15(2):330-40 (2005)) version 1.11 using a set of defaultparameters: -c, --consistency REPS of 2; -ir, --iterative-refinementREPS of 100; -pre, --pre-training REPS of 0. ProbCons is a public domainsoftware program provided by Stanford University.

The default parameters for building an HMM (hmmbuild) are as follows:the default “architecture prior” (archpri) used by MAP architectureconstruction is 0.85, and the default cutoff threshold (idlevel) used todetermine the effective sequence number is 0.62. HMMER 2.3.2 wasreleased October 3, 2003 under a GNU general public license, and isavailable from various sources on the World Wide Web such as hmmerjanelia.org; hmmer wustl.edu; and fr.com/hmmer232/. Hmmbuild outputs themodel as a text file.

The HMM for a group of functional homologs can be used to determine thelikelihood that a candidate cold tolerance-modulating polypeptidesequence is a better fit to that particular HMM than to a null HMMgenerated using a group of sequences that are not structurally orfunctionally related. The likelihood that a candidate polypeptidesequence is a better fit to an HMM than to a null HMM is indicated bythe HMM bit score, a number generated when the candidate sequence isfitted to the HMM profile using the HMMER hmmsearch program. Thefollowing default parameters are used when running hmmsearch: thedefault E-value cutoff (E) is 10.0, the default bit score cutoff (T) isnegative infinity, the default number of sequences in a database (Z) isthe real number of sequences in the database, the default E-value cutofffor the per-domain ranked hit list (domE) is infinity, and the defaultbit score cutoff for the per-domain ranked hit list (domT) is negativeinfinity. A high HMM bit score indicates a greater likelihood that thecandidate sequence carries out one or more of the biochemical orphysiological function(s) of the polypeptides used to generate the HMM.A high HMM bit score is at least 20, and often is higher. Slightvariations in the HMM bit score of a particular sequence can occur dueto factors such as the order in which sequences are processed foralignment by multiple sequence alignment algorithms such as the ProbConsprogram. Nevertheless, such HMM bit score variation is minor

The cold tolerance-modulating polypeptides discussed below fit theindicated HMM with an HMM bit score greater than 20 (e.g., greater than30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500). In someembodiments, the HMM bit score of a cold tolerance-modulatingpolypeptide discussed below is about 50%, 60%, 70%, 80%, 90%, or 95% ofthe HMM bit score of a functional homolog provided in the SequenceListing of this application. In some embodiments, a coldtolerance-modulating polypeptide discussed below fits the indicated HMMwith an HMM bit score greater than 20, and has a domain indicative of acold tolerance-modulating polypeptide. In some embodiments, a coldtolerance-modulating polypeptide discussed below fits the indicated HMMwith an HMM bit score greater than 20, and has 70% or greater sequenceidentity (e.g., 75%, 80%, 85%, 90%, 95%, or 100% sequence identity) toan amino acid sequence shown in any one of FIGS. 1-5.

Examples of polypeptides are shown in the Sequence Listing that have HMMbit scores greater than 130 when fitted to an HMM generated from theamino acid sequences set forth in FIG. 1 are identified in the SequenceListing of this application. Such polypeptides include Ceres CLONE IDno.1915941 (SEQ ID NO: 8), Ceres ANNOT ID no.1461830 (SEQ ID NO: 10),Ceres CLONE ID no.1080942 (SEQ ID NO: 15), and Ceres CLONE ID no.1073190(SEQ ID NO: 17).

Examples of polypeptides are shown in the Sequence Listing that have HMMbit scores greater than 185 when fitted to an HMM generated from theamino acid sequences set forth in FIG. 2 are identified in the SequenceListing of this application. Such polypeptides include Ceres CLONE IDno.1842825 (SEQ ID NO:22), Ceres ANNOT ID no.1482536 (SEQ ID NO: 28),Ceres CLONE ID no.463157 (SEQ ID NO: 32), Ceres CLONE ID no.1674443 (SEQID NO: 40), GI ID no.116310719 (SEQ ID NO: 44), Ceres CLONE ID no.907473(SEQ ID NO: 58), and Ceres CLONE ID no.1755065 (SEQ ID NO:71).

Examples of polypeptides are shown in the Sequence Listing that have HMMbit scores greater than 655 when fitted to an HMM generated from theamino acid sequences set forth in FIG. 3 are identified in the SequenceListing of this application. Such polypeptides include Ceres CLONE IDno.473040 (SEQ ID NO: 79), Ceres CLONE ID no.922223 (SEQ ID NO: 81), GIID no.125528967 (SEQ ID NO: 82), GI ID no.125573200 (SEQ ID NO: 83),Ceres ANNOT ID no.1527409 (SEQ ID NO: 85), GI ID no.92871098 (SEQ ID NO:86), Ceres CLONE ID no.1831117 (SEQ ID NO: 88), and Ceres ANNOT IDno.857222 (SEQ ID NO: 90).

Examples of polypeptides are shown in the Sequence Listing that have HMMbit scores greater than 315 when fitted to an HMM generated from theamino acid sequences set forth in FIG. 4 are identified in the SequenceListing of this application. Such polypeptides include Ceres SEEDLINE IDno.ME17294 (SEQ ID NO: 93), Ceres CLONE ID no.1831117 (SEQ ID NO: 95),Ceres CLONE ID no.1844076 (SEQ ID NO: 98), Ceres CLONE ID no.473040 (SEQID NO: 104), Ceres CLONE ID no.922223 (SEQ ID NO: 106), and GI IDno.125528967 (SEQ ID NO: 107).

Examples of polypeptides are shown in the Sequence Listing that have HMMbit scores greater than 790 when fitted to an HMM generated from theamino acid sequences set forth in FIG. 5 are identified in the SequenceListing of this application. Such polypeptides include LOCUS ID no.AT5G62000 (SEQ ID NO: 112), Ceres ANNOT ID no.1527370 (SEQ ID NO: 114),Ceres ANNOT ID no.1473961 (SEQ ID NO: 116), GI ID no.62319853 (SEQ IDNO: 117), GI ID no.62319903 (SEQ ID NO:118), GI ID no.47716275 (SEQ IDNO: 119), GI ID no.125534572 (SEQ ID NO:120), GI ID no.26251300(SEQ IDNO:121), GI ID no.115441981 (SEQ ID NO:123), GI ID no.23893346 (SEQ IDNO:124), GI ID no.115485689 (SEQ ID NO:125), GI ID no.108864435 (SEQ IDNO:126), GI ID no.50511471 (SEQ ID NO:127), LOCUS ID no. At2g33860 (SEQID NO:128), GI ID no.2245390 (SEQ ID NO:131), GI ID no.3228517 (SEQ IDNO:132), Ceres CLONE ID no.827306 (SEQ ID NO: 134), Ceres CLONE IDno.1598488 (SEQ ID NO: 136), GI ID no.125553314 (SEQ ID NO: 138), andCeres CLONE ID no.462443 (SEQ ID NO:143).

D. Percent Identity

In some embodiments, a cold tolerance-modulating polypeptide has anamino acid sequence with at least 45% sequence identity, e.g., 50%, 52%,56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to one of the amino acid sequences set forth in SEQID NOs: 2, 20, 93, and 74.

Polypeptides having such a percent sequence identity often have a domainindicative of a cold tolerance-modulating polypeptide and/or have an HMMbit score that is greater than 130, as discussed above Amino acidsequences of cold tolerance-modulating polypeptides having at least 80%sequence identity to one of the amino acid sequences set forth in SEQ IDNOs: 2, 20, 93, and 74 are provided in FIGS. 1-5 and in the SequenceListing.

“Percent sequence identity” refers to the degree of sequence identitybetween any given reference sequence, e.g., SEQ ID NO: 2, and acandidate cold tolerance-modulating sequence. A candidate sequencetypically has a length that is from 80 percent to 200 percent of thelength of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97,99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200percent of the length of the reference sequence. A percent identity forany candidate nucleic acid or polypeptide relative to a referencenucleic acid or polypeptide can be determined as follows. A referencesequence (e.g., a nucleic acid sequence or an amino acid sequence) isaligned to one or more candidate sequences using the computer programClustalW (version 1.83, default parameters), which allows alignments ofnucleic acid or polypeptide sequences to be carried out across theirentire length (global alignment). Chenna et al., Nucleic Acids Res.,31(13):3497-500 (2003).

ClustalW calculates the best match between a reference and one or morecandidate sequences, and aligns them so that identities, similaritiesand differences can be determined. Gaps of one or more residues can beinserted into a reference sequence, a candidate sequence, or both, tomaximize sequence alignments. For fast pairwise alignment of nucleicacid sequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The ClustalW output is asequence alignment that reflects the relationship between sequences.ClustalW can be run, for example, at the Baylor College of MedicineSearch Launcher site(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at theEuropean Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

To determine percent identity of a candidate nucleic acid or amino acidsequence to a reference sequence, the sequences are aligned usingClustalW, the number of identical matches in the alignment is divided bythe length of the reference sequence, and the result is multiplied by100. It is noted that the percent identity value can be rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded upto 78.2.

In some cases, a cold tolerance-modulating polypeptide has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 2. Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 2 are provided in FIG. 1 andin the Sequence Listing. Examples of such polypeptides include CeresCLONE ID no.1915941 (SEQ ID NO: 8), Ceres ANNOT ID no.1461830 (SEQ IDNO: 10), Ceres CLONE ID no.1080942 (SEQ ID NO: 15), and Ceres CLONE IDno.1073190 (SEQ ID NO: 17).

In some cases, a cold tolerance-modulating polypeptide has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 20 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 20 are provided in FIG. 2 andin the Sequence Listing. Examples of such polypeptides include CeresCLONE ID no.1842825 (SEQ ID NO: 22), Ceres ANNOT ID no.1482536 (SEQ IDNO: 28), Ceres CLONE ID no.463157 (SEQ ID NO: 32), Ceres CLONE IDno.1674443 (SEQ ID NO:40), GI ID no.116310719 (SEQ ID NO: 44), CeresCLONE ID no.907473 (SEQ ID NO: 58), and Ceres CLONE ID no.1755065 (SEQID NO: 71).

In some cases, a cold tolerance-modulating polypeptide has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 93 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 93 are provided in FIG. 3 andin the Sequence Listing. Examples of such polypeptides include CeresCLONE ID no.473040 (SEQ ID NO: 79), Ceres CLONE ID no.922223 (SEQ ID NO:81), GI ID no.125528967, (SEQ ID NO: 82), GI ID no.125573200 (SEQ ID NO:83), Ceres ANNOT ID no.1527409 (SEQ ID NO: 85), GI ID no.92871098 (SEQID NO: 86), Ceres CLONE ID no.1831117 (SEQ ID NO: 88), and Ceres ANNOTID no.857222 (SEQ ID NO: 90).

In some cases, a cold tolerance-modulating polypeptide has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 93 Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 93 are provided in FIG. 4 andin the Sequence Listing. Examples of such polypeptides include CeresCLONE ID no.1831117 (SEQ ID NO: 95), Ceres CLONE ID no.1844076 (SEQ IDNO: 98), Ceres CLONE ID no.473040 (SEQ ID NO: 104), Ceres CLONE IDno.922223 (SEQ ID NO: 106), and GI ID no.125528967 (SEQ ID NO: 107).

In some cases, a cold tolerance-modulating tasiRNA acts upon an ARFamino acid sequence with at least 45% sequence identity, e.g., 50%, 52%,56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to the amino acid sequence set forth in SEQ ID NO:116 Amino acid sequences of polypeptides having greater than 45%sequence identity to the polypeptide set forth in SEQ ID NO: 116 areprovided in FIG. 5 and in the Sequence Listing. Examples of suchpolypeptides include LOCUS ID no. AT5G62000 (SEQ ID NO: 112), CeresANNOT ID no.1527370 (SEQ ID NO: 114), GI ID no.62319853 (SEQ ID NO:117), GI ID no.62319903 (SEQ ID NO:118), GI ID no.47716275 (SEQ ID NO:119), GI ID no.125534572 (SEQ ID NO:120), GI ID no.26251300(SEQ IDNO:121), GI ID no.115441981 (SEQ ID NO:123), GI ID no.23893346 (SEQ IDNO:124), GI ID no.115485689 (SEQ ID NO:125), GI ID no.108864435 (SEQ IDNO:126), GI ID no.50511471 (SEQ ID NO:127), LOCUS ID no. At2g33860 (SEQID NO:128), GI ID no.2245390 (SEQ ID NO:131), GI ID no.3228517 (SEQ IDNO:132), Ceres CLONE ID no.827306 (SEQ ID NO: 134), Ceres CLONE IDno.1598488 (SEQ ID NO: 136), GI ID no.125553314 (SEQ ID NO: 138), andCeres CLONE ID no.462443 (SEQ ID NO:143).

E. Other Sequences

It should be appreciated that a cold tolerance-modulating polypeptidecan include additional amino acids that are not involved in coldtolerance modulation, and thus such a polypeptide can be longer thanwould otherwise be the case. For example, a cold tolerance-modulatingpolypeptide can include a purification tag, a chloroplast transitpeptide, a mitochondrial transit peptide, an amyloplast transit peptide,a lysosome signal peptide or a leader sequence added to the amino orcarboxy terminus. In some embodiments, a cold tolerance-modulatingpolypeptide includes an amino acid sequence that functions as areporter, e.g., a green fluorescent protein or yellow fluorescentprotein.

III. Nucleic Acids

Nucleic acids described herein include nucleic acids that are effectiveto modulate cold tolerance levels when transcribed in a plant or plantcell. Such nucleic acids include, without limitation, those that encodea cold tolerance-modulating polypeptide and those that can be used toinhibit expression of a cold tolerance-modulating polypeptide via anucleic acid based method.

A. Cold Tolerance-Modulating Nucleic Acids

Nucleic acids encoding cold tolerance-modulating polypeptides aredescribed herein. Examples of such nucleic acids include SEQ ID NOs: 3,5, 7, 9, 11, 14, 16, 18, 19, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41, 55,57, 70, 75, 78, 80, 84, 87, 89, 91, 92, 94, 97, 99, 103, 105, 109, 113,115, 129, 133, 135, 140, and 142, as described in more detail below. Anucleic acid also can be a fragment that is at least 40% (e.g., at least45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%) of the length of thefull-length nucleic acid set forth in SEQ ID NOs: 3, 5, 7, 9, 11, 14,16, 18, 19, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41, 55, 57, 70, 75, 78,80, 84, 87, 89, 91, 92, 94, 97, 99, 103, 105, 109, 113, 115, 129, 133,135, 140, and 142 or of the length of the full-length nucleic acid setforth in the sequence listing identified as functional homologs of thesequences of FIG. 3.

A cold tolerance-modulating nucleic acid can comprise the nucleotidesequence set forth in SEQ ID NO: 1. Alternatively, a coldtolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 1. For example, acold tolerance-modulating nucleic acid can have a nucleotide sequencewith at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%,or 99% sequence identity, to the nucleotide sequence set forth in SEQ IDNO: 1.

A cold tolerance-modulating nucleic acid can comprise the nucleotidesequence set forth in SEQ ID NO: 19. Alternatively, a coldtolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 19. For example,a cold tolerance-modulating nucleic acid can have a nucleotide sequencewith at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%,or 99% sequence identity, to the nucleotide sequence set forth in SEQ IDNO: 19.

A cold tolerance-modulating nucleic acid can comprise the nucleotidesequence set forth in SEQ ID NO: 92. Alternatively, a coldtolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 92. For example,a cold tolerance-modulating nucleic acid can have a nucleotide sequencewith at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%,or 99% sequence identity, to the nucleotide sequence set forth in SEQ IDNO: 92.

A cold tolerance-modulating nucleic acid can comprise the nucleotidesequence set forth in SEQ ID NO: 97. Alternatively, a coldtolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 97. For example,a cold tolerance-modulating nucleic acid can have a nucleotide sequencewith at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%,or 99% sequence identity, to the nucleotide sequence set forth in SEQ IDNO: 97.

A cold tolerance-modulating nucleic acid can comprise the nucleotidesequence set forth in SEQ ID NO: 111. Alternatively, a coldtolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 111. For example,a cold tolerance-modulating nucleic acid can have a nucleotide sequencewith at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%,or 99% sequence identity, to the nucleotide sequence set forth in SEQ IDNO: 111.

A cold tolerance-modulating sequence can be at least a fragment of anucleotide sequence such as Ceres ANNOT ID no.1473961 (SEQ ID NO: 116)or homologs thereof. For example, the cold tolerance-modulatingnucleotide may be a tasiRNA. Such cold tolerance-modulating nucleotidesequences can act upon a protein that comprises an auxin response factormotif. This motif is present in SEQ ID NO: 112, which sets forth theamino acid sequence of an Arabidopsis clone, identified herein as LOCUSID no. AT5G62000 (SEQ ID NO: 112), that is predicted to encode anpolypeptide comprising an auxin response factor motif. In certainembodiments, the protein comprising an auxin response factor motif is anARF protein. The ARFs are key regulators of auxin-modulated geneexpression. There are multiple ARF proteins, some of which activate,while others repress transcription. ARF proteins bind toauxin-responsive cis-acting promoter elements (AuxREs) using anN-terminal DNA-binding domain. It is thought that Aux/IAA proteinsactivate transcription by modifying ARF activity through the C-terminalprotein-protein interaction domains found in both Aux/IAA and ARFproteins.

A cold tolerance-modulating sequence can be at least a fragment of anucleotide sequence such as Ceres ANNOT ID no.1473961 (SEQ ID NO: 116)or homologs thereof. For example, the cold tolerance-modulatingnucleotide may be a tasiRNA. Such cold tolerance-modulating nucleotidesequences can act upon a protein that comprises a B3 DNA binding domain.This domain is present in SEQ ID NO: 112, which sets forth the aminoacid sequence of an Arabidopsis clone, identified herein as LOCUS ID no.AT5G62000 (SEQ ID NO: 112), that is predicted to encode an polypeptidecomprising an B3 DNA binding domain. In certain embodiments, the proteincomprising a B3 DNA binding domain is an ARF protein.

In some embodiments, a cold tolerance-modulating sequence is a tasiRNAsequence or a homolog thereof, such tasiRNA sequence being encoded by anucleic acid sequence that comprises a domain having 80% or greatersequence identity to nucleic acid residues 305 to about 346 of SEQ IDNO: 111, residues 21 to about 62 of SEQ ID NO: 66, residues 20 to about61 of SEQ ID NO: 67, residues 21 to about 62 of SEQ ID NO: 72, residues21 to about 62 of SEQ ID NO: 73, residues 77 to about 118 of SEQ ID NO:144, residues 292 to about 313 of SEQ ID NO: 145, residues 37 to about78 of SEQ ID NO: 146, residues 56 to about 97 of SEQ ID NO: 147,residues 37 to about 78 of SEQ ID NO: 148, residues 45 to about 86 ofSEQ ID NO: 149, residues 46 to about 98 of SEQ ID NO: 150, residues 476to about 497 of SEQ ID NO: 151, residues 21 to about 62 of SEQ ID NO:152, residues 21 to about 62 of SEQ ID NO: 153, residues 21 to about 62of SEQ ID NO: 154, residues 21 to about 62 of SEQ ID NO: 155, orresidues 21 to about 62 of SEQ ID NO: 156.

In some embodiments, a cold tolerance-modulating sequence is anucleotide sequence or a homolog thereof, such as a tasiRNA sequence,wherein said nucleotide is encoded by a nucleic acid sequence that alsocomprises an miR390 recognition sequence having 80% or greater sequenceidentity to nucleic acid residues 109 to about 129 of SEQ ID NO: 66,residues 114 to about 135 of SEQ ID NO: 67, residues 119 to about 139 ofSEQ ID NO: 72, residues 108 to about 128 of SEQ ID NO: 73, residues 234to about 254 of SEQ ID NO: 144, residues 135 to about 176 of SEQ ID NO:145, residues 173 to about 189 of SEQ ID NO: 147, residues 154 to about170 of SEQ ID NO: 148, residues 134 to about 157 of SEQ ID NO: 149,residues 154 to about 198 of SEQ ID NO: 150, residues 319 to about 360of SEQ ID NO: 151, residues 121 to about 141 of SEQ ID NO: 152, residues120 to about 140 of SEQ ID NO: 153, residues 121 to about 141 of SEQ IDNO: 154, residues 121 to about 141 of SEQ ID NO: 155, residues 121 toabout 141 of SEQ ID NO: 156, or residues 462 to about 483 of SEQ ID NO:111. miR390 recognition sequences may guide in-phase processing oftranscription (Allen et al. 2005).

In embodiments of the invention, a cold tolerance-modulating nucleotide,such as Ceres ANNOT ID no.1473961 (SEQ ID NO: 116) or a homolog threof,can act upon an polypeptide that comprises a B3 DNA binding domainhaving 80% or greater sequence identity to amino acid residues 163 to268 of SEQ ID NO: 112, residues 157 to 262 of SEQ ID NO: 114, residues157 to 262 of SEQ ID NO: 116, residues 163 to 268 of SEQ ID NO: 117,residues 163 to 268 of SEQ ID NO: 118, residues 158 to 263 of SEQ ID NO:119, residues 148 to 253 of SEQ ID NO: 120, residues 147 to 252 of SEQID NO: 121, residues 123 to 228 of SEQ ID NO: 122, residues 128 to 233of SEQ ID NO: 123, residues 131 to 236 of SEQ ID NO: 124, residues 147to 252 of SEQ ID NO: 125, residues 148 to 253 of SEQ ID NO: 126,residues 141 to 246 of SEQ ID NO: 127, residues 158 to 263 of SEQ ID NO:128, residues 142 to 247 of SEQ ID NO: 130, residues 158 to 263 of SEQID NO: 131, residues 158 to 263 of SEQ ID NO: 132, residues 126 to 231of SEQ ID NO: 134, residues 129 to 234 of SEQ ID NO: 136, residues 114to 219 of SEQ ID NO: 137, residues 141 to 246 of SEQ ID NO: 138,residues 176 to 281 of SEQ ID NO: 139, residues 152 to 257 of SEQ ID NO:141, or residues 121 to 225 of SEQ ID NO: 143.

In embodiments of the invention, a cold tolerance-modulating tasiRNAsequence such as Ceres ANNOT ID no.1473961 (SEQ ID NO: 116) can act uponan ARF polypeptide that comprises an auxin response factor motif having80% or greater sequence identity to amino acid residues 290 to 372 ofSEQ ID NO: 112, residues 284 to 366 of SEQ ID NO: 114, residues 284 to366 of SEQ ID NO: 116, residues 290 to 372 of SEQ ID NO: 117, residues290 to 372 of SEQ ID NO: 118, residues 285 to 367 of SEQ ID NO: 119,residues 275 to 357 of SEQ ID NO: 120, residues 274 to 356 of SEQ ID NO:121, residues 250 to 331 of SEQ ID NO: 122, residues 255 to 336 of SEQID NO: 123, residues 258 to 340 of SEQ ID NO: 124, residues 274 to 356of SEQ ID NO: 125, residues 275 to 357 of SEQ ID NO: 126, residues 268to 349 of SEQ ID NO: 127, residues 285 to 367 of SEQ ID NO: 128,residues 269 to 351 of SEQ ID NO: 130, residues 285 to 367 of SEQ ID NO:131, residues 285 to 367 of SEQ ID NO: 132, residues 253 to 334 of SEQID NO: 134, residues 256 to 337 of SEQ ID NO: 136, residues 241 to 322of SEQ ID NO: 137, residues 268 to 349 of SEQ ID NO: 138, residues 302to 384 of SEQ ID NO: 139, residues 279 to 361 of SEQ ID NO: 141, orresidues 247 to 332 of SEQ ID NO: 143.

Isolated nucleic acid molecules can be produced by standard techniques.For example, polymerase chain reaction (PCR) techniques can be used toobtain an isolated nucleic acid containing a nucleotide sequencedescribed herein. PCR can be used to amplify specific sequences from DNAas well as RNA, including sequences from total genomic DNA or totalcellular RNA. Various PCR methods are described, for example, in PCRPrimer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold SpringHarbor Laboratory Press, 1995. Generally, sequence information from theends of the region of interest or beyond is employed to designoligonucleotide primers that are identical or similar in sequence toopposite strands of the template to be amplified. Various PCR strategiesalso are available by which site-specific nucleotide sequencemodifications can be introduced into a template nucleic acid. Isolatednucleic acids also can be chemically synthesized, either as a singlenucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector. Isolated nucleic acids of the invention also canbe obtained by mutagenesis of, e.g., a naturally occurring DNA.

B. Use of Nucleic Acids to Modulate Expression of Polypeptides

i. Expression of a Cold Tolerance-Modulating Polypeptide

A nucleic acid encoding one of the cold tolerance-modulatingpolypeptides described herein can be used to express the polypeptide ina plant species of interest, typically by transforming a plant cell witha nucleic acid having the coding sequence for the polypeptide operablylinked in sense orientation to one or more regulatory regions. It willbe appreciated that because of the degeneracy of the genetic code, anumber of nucleic acids can encode a particular coldtolerance-modulating polypeptide; i.e., for many amino acids, there ismore than one nucleotide triplet that serves as the codon for the aminoacid. Thus, codons in the coding sequence for a given coldtolerance-modulating polypeptide can be modified such that optimalexpression in a particular plant species is obtained, using appropriatecodon bias tables for that species.

In some cases, expression of a cold tolerance-modulating polypeptideinhibits one or more functions of an endogenous polypeptide. Forexample, a nucleic acid that encodes a dominant negative polypeptide canbe used to inhibit protein function. A dominant negative polypeptidetypically is mutated or truncated relative to an endogenous wild typepolypeptide, and its presence in a cell inhibits one or more functionsof the wild type polypeptide in that cell, i.e., the dominant negativepolypeptide is genetically dominant and confers a loss of function. Themechanism by which a dominant negative polypeptide confers such aphenotype can vary but often involves a protein-protein interaction or aprotein-DNA interaction. For example, a dominant negative polypeptidecan be an enzyme that is truncated relative to a native wild typeenzyme, such that the truncated polypeptide retains domains involved inbinding a first protein but lacks domains involved in binding a secondprotein. The truncated polypeptide is thus unable to properly modulatethe activity of the second protein. See, e.g., US 2007/0056058. Asanother example, a point mutation that results in a non-conservativeamino acid substitution in a catalytic domain can result in a dominantnegative polypeptide. See, e.g., US 2005/032221. As another example, adominant negative polypeptide can be a transcription factor that istruncated relative to a native wild type transcription factor, such thatthe truncated polypeptide retains the DNA binding domain(s) but lacksthe activation domain(s). Such a truncated polypeptide can inhibit thewild type transcription factor from binding DNA, thereby inhibitingtranscription activation.

ii. Inhibition of Expression of a Cold Tolerance-Modulating Polypeptide

Polynucleotides and recombinant constructs described herein can be usedto inhibit expression of a cold tolerance-modulating polypeptide in aplant species of interest. See, e.g., Matzke and Birchler, NatureReviews Genetics 6:24-35 (2005); Akashi et al., Nature Reviews Mol. CellBiology 6:413-422 (2005); Mittal, Nature Reviews Genetics 5:355-365(2004); Dorsett and Tuschl, Nature Reviews Drug Discovery 3: 318-329(2004); and Nature Reviews RNA interference collection, October 2005 atnature.com/reviews/focus/mai. Typically, at least a fragment of anucleic acid encoding cold tolerance-modulating polypeptides and/or itscomplement is expressed. A fragment is typically at least 20 nucleotideslong, as needed for the methods noted below. A number of nucleic acidbased methods, including antisense RNA, ribozyme directed RNA cleavage,post-transcriptional gene silencing (PTGS), e.g., RNA interference(RNAi), and transcriptional gene silencing (TGS) are known to inhibitgene expression in plants. Suitable polynucleotides include full-lengthnucleic acids encoding cold tolerance-modulating polypeptides orfragments of such full-length nucleic acids. In some embodiments, acomplement of the full-length nucleic acid or a fragment thereof can beused. Typically, a fragment is at least 10 nucleotides, e.g., at least12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 35,40, 50, 80, 100, 200, 500 nucleotides or more. Generally, higherhomology can be used to compensate for the use of a shorter sequence.

Antisense technology is one well-known method. In this method, a nucleicacid segment from a gene to be repressed is cloned and operably linkedto a regulatory region and a transcription termination sequence so thatthe antisense strand of RNA is transcribed. The recombinant construct isthen transformed into plants, as described herein, and the antisensestrand of RNA is produced. The nucleic acid segment need not be theentire sequence of the gene to be repressed, but typically will besubstantially complementary to at least a portion of the sense strand ofthe gene to be repressed. Generally, higher homology can be used tocompensate for the use of a shorter sequence. Typically, a sequence ofat least 30 nucleotides is used, e.g., at least 40, 50, 80, 100, 200,500 nucleotides or more.

In another method, a nucleic acid can be transcribed into a ribozyme, orcatalytic RNA, that affects expression of an mRNA. See, U.S. Pat. No.6,423,885. Ribozymes can be designed to specifically pair with virtuallyany target RNA and cleave the phosphodiester backbone at a specificlocation, thereby functionally inactivating the target RNA. Heterologousnucleic acids can encode ribozymes designed to cleave particular mRNAtranscripts, thus preventing expression of a polypeptide. Hammerheadribozymes are useful for destroying particular mRNAs, although variousribozymes that cleave mRNA at site-specific recognition sequences can beused. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target RNA contains a 5′-UG-3′nucleotide sequence. The construction and production of hammerheadribozymes is known in the art. See, for example, U.S. Pat. No. 5,254,678and WO 02/46449 and references cited therein. Hammerhead ribozymesequences can be embedded in a stable RNA such as a transfer RNA (tRNA)to increase cleavage efficiency in vivo. Perriman et al., Proc. Natl.Acad. Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methodsin Molecular Biology, Vol. 74, Chapter 43, “Expressing Ribozymes inPlants”, Edited by Turner, P. C., Humana Press Inc., Totowa, N.J. RNAendoribonucleases which have been described, such as the one that occursnaturally in Tetrahymena thermophila, can be useful. See, for example,U.S. Pat. Nos. 4,987,071 and 6,423,885.

PTGS, e.g., RNAi, can also be used to inhibit the expression of a gene.For example, a construct can be prepared that includes a sequence thatis transcribed into an RNA that can anneal to itself, e.g., a doublestranded RNA having a stem-loop structure. In some embodiments, onestrand of the stem portion of a double stranded RNA comprises a sequencethat is similar or identical to the sense coding sequence or a fragmentthereof of a cold tolerance-modulating polypeptide, and that is fromabout 10 nucleotides to about 2,500 nucleotides in length. The length ofthe sequence that is similar or identical to the sense coding sequencecan be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25nucleotides to 100 nucleotides. The other strand of the stem portion ofa double stranded RNA comprises a sequence that is similar or identicalto the antisense strand or a fragment thereof of the coding sequence ofthe cold tolerance-modulating polypeptide, and can have a length that isshorter, the same as, or longer than the corresponding length of thesense sequence. In some cases, one strand of the stem portion of adouble stranded RNA comprises a sequence that is similar or identical tothe 3′ or 5′ untranslated region, or a fragment thereof, of an mRNAencoding a cold tolerance-modulating polypeptide, and the other strandof the stem portion of the double stranded RNA comprises a sequence thatis similar or identical to the sequence that is complementary to the 3′or 5′ untranslated region, respectively, or a fragment thereof, of themRNA encoding the cold tolerance-modulating polypeptide. In otherembodiments, one strand of the stem portion of a double stranded RNAcomprises a sequence that is similar or identical to the sequence of anintron, or a fragment thereof, in the pre-mRNA encoding a coldtolerance-modulating polypeptide, and the other strand of the stemportion comprises a sequence that is similar or identical to thesequence that is complementary to the sequence of the intron, or afragment thereof, in the pre-mRNA. The loop portion of a double strandedRNA can be from 3 nucleotides to 5,000 nucleotides, e.g., from 3nucleotides to 25 nucleotides, from 15 nucleotides to 1,000 nucleotides,from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200nucleotides. The loop portion of the RNA can include an intron. A doublestranded RNA can have zero, one, two, three, four, five, six, seven,eight, nine, ten, or more stem-loop structures. A construct including asequence that is operably linked to a regulatory region and atranscription termination sequence, and that is transcribed into an RNAthat can form a double stranded RNA, is transformed into plants asdescribed herein. Methods for using RNAi to inhibit the expression of agene are known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588.See also WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S.Patent Publications 20030175965, 20030175783, 20040214330, and20030180945.

microRNA (miRNA) and tasiRNA, which are non-protein coding RNAs, canalso be used to inhibit the expression of a gene. The gene targeted forinhibition may be an endogenous plant gene, a viral gene, a bacterialgene, a fungal gene, or an insect gene. miRNAs and tasiRNAs areregulatory agents consisting of about 19 to 25 ribonucleotides. miRNAare highly efficient at inhibiting the expression of endogenous genesand/or can guide in-phase processing of tasiRNA primary transcripts.tasiRNAs similarly inhibit gene expression by interacting with targetmRNAs and guide cleavage by the same mechanism as do plant miRNAs, butdiffer from miRNAs in that they arise from double-stranded RNA, whichmay require RNA-dependent RNA polymerases.

For example, a tasiRNA can act upon an auxin responsive protein (ARF)(e.g., ARF3 or ARF4). In particular, inhibition of the expression of anARF encoding gene (e.g., ARF3 or ARF4) may be obtained by interferenceby expression of a nucleic acid sequence encoding a tasiRNA.Transcription of a tasiRNA encoding nucleic acid sequence can be underthe control of a promoter, such as, but not limited to, those promotersand regulatory regions described herein, or under promotional control ofa tasiRNA coding sequence's own promoter. For such interference, theexpression cassette is designed to express an RNA molecule that ismodeled on an endogenous tasiRNA encoding sequence. The tasiRNA encodingsequence encodes an RNA that forms a hairpin structure containing anucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of ARF protein expression, thenucleotide sequence is selected from an ARF transcript sequence andcontains about 19 to 25 nucleotides of said ARF protein sequence insense orientation and about 19 to 25 nucleotides of a correspondingantisense sequence that is complementary to the sense sequence. tasiRNAmolecules are highly efficient at inhibiting the expression ofendogenous genes. In Arabidopsis, a nuclear DCL enzyme is believed to berequired for mature miRNA formation (Xie et al. (2004) PLoS Biol.,2:642-652, which is incorporated by reference herein) Inhibition of geneexpression by miRNAs and tasiRNAs and methods for inhibition are knownto those of skill in the art. See, for example, Javier, et al., (2003)Nature 425:257-263; Bartel (2004) Cell, 116:281-297; Kim (2005) NatureRev. Mol. Cell Biol., 6:376-385; and Allen et al. (2005) Cell,121:207-221, all of which are incorporated by reference herein.

Constructs containing regulatory regions operably linked to nucleic acidmolecules in sense orientation can also be used to inhibit theexpression of a gene. The transcription product can be similar oridentical to the sense coding sequence, or a fragment thereof, of a coldtolerance-modulating polypeptide. The transcription product also can beunpolyadenylated, lack a 5′ cap structure, or contain an unspliceableintron. Methods of inhibiting gene expression using a full-length cDNAas well as a partial cDNA sequence are known in the art. See, e.g., U.S.Pat. No. 5,231,020.

In some embodiments, a construct containing a nucleic acid having atleast one strand that is a template for both sense and antisensesequences that are complementary to each other is used to inhibit theexpression of a gene. The sense and antisense sequences can be part of alarger nucleic acid molecule or can be part of separate nucleic acidmolecules having sequences that are not complementary. The sense orantisense sequence can be a sequence that is identical or complementaryto the sequence of an mRNA, the 3′ or 5′ untranslated region of an mRNA,or an intron in a pre-mRNA encoding a cold tolerance-modulatingpolypeptide, or a fragment of such sequences. In some embodiments, thesense or antisense sequence is identical or complementary to a sequenceof the regulatory region that drives transcription of the gene encodinga cold tolerance-modulating polypeptide. In each case, the sensesequence is the sequence that is complementary to the antisensesequence.

The sense and antisense sequences can be any length greater than about10 nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, or more nucleotides). For example, anantisense sequence can be 21 or 22 nucleotides in length. Typically, thesense and antisense sequences range in length from about 15 nucleotidesto about 30 nucleotides, e.g., from about 18 nucleotides to about 28nucleotides, or from about 21 nucleotides to about 25 nucleotides.

In some embodiments, an antisense sequence is a sequence complementaryto an mRNA sequence, or a fragment thereof, encoding a coldtolerance-modulating polypeptide described herein. The sense sequencecomplementary to the antisense sequence can be a sequence present withinthe mRNA of the cold tolerance-modulating polypeptide. Typically, senseand antisense sequences are designed to correspond to a 15-30 nucleotidesequence of a target mRNA such that the level of that target mRNA isreduced.

In some embodiments, a construct containing a nucleic acid having atleast one strand that is a template for more than one sense sequence(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sense sequences) can be usedto inhibit the expression of a gene. Likewise, a construct containing anucleic acid having at least one strand that is a template for more thanone antisense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreantisense sequences) can be used to inhibit the expression of a gene.For example, a construct can contain a nucleic acid having at least onestrand that is a template for two sense sequences and two antisensesequences. The multiple sense sequences can be identical or different,and the multiple antisense sequences can be identical or different. Forexample, a construct can have a nucleic acid having one strand that is atemplate for two identical sense sequences and two identical antisensesequences that are complementary to the two identical sense sequences.Alternatively, an isolated nucleic acid can have one strand that is atemplate for (1) two identical sense sequences 20 nucleotides in length,(2) one antisense sequence that is complementary to the two identicalsense sequences 20 nucleotides in length, (3) a sense sequence 30nucleotides in length, and (4) three identical antisense sequences thatare complementary to the sense sequence 30 nucleotides in length. Theconstructs provided herein can be designed to have any arrangement ofsense and antisense sequences. For example, two identical sensesequences can be followed by two identical antisense sequences or can bepositioned between two identical antisense sequences.

A nucleic acid having at least one strand that is a template for one ormore sense and/or antisense sequences can be operably linked to aregulatory region to drive transcription of an RNA molecule containingthe sense and/or antisense sequence(s). In addition, such a nucleic acidcan be operably linked to a transcription terminator sequence, such asthe terminator of the nopaline synthase (nos) gene. In some cases, tworegulatory regions can direct transcription of two transcripts: one fromthe top strand, and one from the bottom strand. See, for example, Yan etal., Plant Physiol., 141:1508-1518 (2006). The two regulatory regionscan be the same or different. The two transcripts can formdouble-stranded RNA molecules that induce degradation of the target RNA.In some cases, a nucleic acid can be positioned within a T-DNA orplant-derived transfer DNA (P-DNA) such that the left and right T-DNAborder sequences, or the left and right border-like sequences of theP-DNA, flank or are on either side of the nucleic acid. See, US2006/0265788. The nucleic acid sequence between the two regulatoryregions can be from about 15 to about 300 nucleotides in length. In someembodiments, the nucleic acid sequence between the two regulatoryregions is from about 15 to about 200 nucleotides in length, from about15 to about 100 nucleotides in length, from about 15 to about 50nucleotides in length, from about 18 to about 50 nucleotides in length,from about 18 to about 40 nucleotides in length, from about 18 to about30 nucleotides in length, or from about 18 to about 25 nucleotides inlength.

In some nucleic-acid based methods for inhibition of gene expression inplants, a suitable nucleic acid can be a nucleic acid analog. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered morpholino ring, or peptidenucleic acids, in which the deoxyphosphate backbone is replaced by apseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev.,7:187-195; Hyrup et al., Bioorgan. Med. Chem., 4:5-23 (1996). Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

C. Constructs/Vectors

Recombinant constructs provided herein can be used to transform plantsor plant cells in order to modulate cold tolerance levels. A recombinantnucleic acid construct can comprise a nucleic acid encoding a coldtolerance-modulating polypeptide as described herein, operably linked toa regulatory region suitable for expressing the coldtolerance-modulating polypeptide in the plant or cell. Thus, a nucleicacid can comprise a coding sequence that encodes any of the coldtolerance-modulating polypeptides as set forth in SEQ ID NOs: 2, 20, 93,74, or a homologs thereof. Examples of nucleic acids encoding coldtolerance-modulating polypeptides are set forth in SEQ ID NOs: 1, 19,92, 97, or 111, and in FIGS. 1-5 and in the Sequence Listing. The coldtolerance-modulating polypeptide encoded by a recombinant nucleic acidcan be a native cold tolerance-modulating polypeptide, or can beheterologous to the cell. In some cases, the recombinant constructcontains a nucleic acid that inhibits expression of a coldtolerance-modulating polypeptide, operably linked to a regulatoryregion. Examples of suitable regulatory regions are described in thesection entitled “Regulatory Regions.”

Vectors containing recombinant nucleic acid constructs such as thosedescribed herein also are provided. Suitable vector backbones include,for example, those routinely used in the art such as plasmids, viruses,artificial chromosomes, BACs, YACs, or PACs. Suitable expression vectorsinclude, without limitation, plasmids and viral vectors derived from,for example, bacteriophage, baculoviruses, and retroviruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

The vectors provided herein also can include, for example, origins ofreplication, scaffold attachment regions (SARs), and/or markers. Amarker gene can confer a selectable phenotype on a plant cell. Forexample, a marker can confer biocide resistance, such as resistance toan antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or anherbicide (e.g., glyphosate, chlorsulfuron or phosphinothricin). Inaddition, an expression vector can include a tag sequence designed tofacilitate manipulation or detection (e.g., purification orlocalization) of the expressed polypeptide. Tag sequences, such asluciferase, β-glucuronidase (GUS), green fluorescent protein (GFP),glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, orFlag™ tag (Kodak, New Haven, Conn.) sequences typically are expressed asa fusion with the encoded polypeptide. Such tags can be insertedanywhere within the polypeptide, including at either the carboxyl oramino terminus.

D. Regulatory Regions

The choice of regulatory regions to be included in a recombinantconstruct depends upon several factors, including, but not limited to,efficiency, selectability, inducibility, desired expression level, andcell- or tissue-preferential expression. It is a routine matter for oneof skill in the art to modulate the expression of a coding sequence byappropriately selecting and positioning regulatory regions relative tothe coding sequence. Transcription of a nucleic acid can be modulated ina similar manner

Some suitable regulatory regions initiate transcription only, orpredominantly, in certain cell types. Methods for identifying andcharacterizing regulatory regions in plant genomic DNA are known,including, for example, those described in the following references:Jordano et al., Plant Cell, 1:855-866 (1989); Bustos et al., Plant Cell,1:839-854 (1989); Green et al., EMBO J., 7:4035-4044 (1988); Meier etal., Plant Cell, 3:309-316 (1991); and Zhang et al., Plant Physiology,110:1069-1079 (1996).

Examples of various classes of regulatory regions are described below.Some of the regulatory regions indicated below as well as additionalregulatory regions are described in more detail in U.S. PatentApplication Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869;60/583,691; 60/619,181; 60/637,140; 60/757,544; 60/776,307; 10/957,569;11/058,689; 11/172,703; 11/208,308; 11/274,890; 60/583,609; 60/612,891;11/097,589; 11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017;PCT/US05/011105; PCT/US05/23639; PCT/US05/034308; PCT/US05/034343; andPCT/US06/038236; PCT/US06/040572; and PCT/US07/62762.

For example, the sequences of regulatory regions p326, YP0144, YP0190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633,YP0128, YP0275, PT0660, PT0683, PT0758, PT0613, PT0672, PT0688, PT0837,YP0092, PT0676, PT0708, YP0396, YP0007, YP0111, YP0103, YP0028, YP0121,YP0008, YP0039, YP0115, YP0119, YP0120, YP0374, YP0101, YP0102, YP0110,YP0117, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143, YP0156,PT0650, PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886,PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286, YP0377,PD1367, PT0863, PT0829, PT0665, PT0678, YP0086, YP0188, YP0263, PT0743and YP0096 are set forth in the sequence listing of PCT/US06/040572; thesequence of regulatory region PT0625 is set forth in the sequencelisting of PCT/US05/034343; the sequences of regulatory regions PT0623,YP0388, YP0087, YP0093, YP0108, YP0022 and YP0080 are set forth in thesequence listing of U.S. patent application Ser. No. 11/172,703; thesequence of regulatory region PR0924 is set forth in the sequencelisting of PCT/US07/62762; and the sequences of regulatory regionsp530c10, pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285 are set forth in thesequence listing of PCT/US06/038236.

It will be appreciated that a regulatory region may meet criteria forone classification based on its activity in one plant species, and yetmeet criteria for a different classification based on its activity inanother plant species.

ii Broadly Expressing Promoters

A promoter can be said to be “broadly expressing” when it promotestranscription in many, but not necessarily all, plant tissues. Forexample, a broadly expressing promoter can promote transcription of anoperably linked sequence in one or more of the shoot, shoot tip (apex),and leaves, but weakly or not at all in tissues such as roots or stems.As another example, a broadly expressing promoter can promotetranscription of an operably linked sequence in one or more of the stem,shoot, shoot tip (apex), and leaves, but can promote transcriptionweakly or not at all in tissues such as reproductive tissues of flowersand developing seeds. Non-limiting examples of broadly expressingpromoters that can be included in the nucleic acid constructs providedherein include the p326, YP0144, YP0190, p13879, YP0050, p32449, 21876,YP0158, YP0214, YP0380, PT0848, and PT0633 promoters. Additionalexamples include the cauliflower mosaic virus (CaMV) 35S promoter, themannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived fromT-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34Spromoter, actin promoters such as the rice actin promoter, and ubiquitinpromoters such as the maize ubiquitin-1 promoter. In some cases, theCaMV 35S promoter is excluded from the category of broadly expressingpromoters.

ii. Root Promoters

Root-active promoters confer transcription in root tissue, e.g., rootendodermis, root epidermis, or root vascular tissues. In someembodiments, root-active promoters are root-preferential promoters,i.e., confer transcription only or predominantly in root tissue.Root-preferential promoters include the YP0128, YP0275, PT0625, PT0660,PT0683, and PT0758 promoters. Other root-preferential promoters includethe PT0613, PT0672 , PT0688, and PT0837 promoters, which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV 35S promoter (Lam et al., Proc.Natl. Acad. Sci. USA, 86:7890-7894 (1989)), root cell specific promotersreported by Conkling et al., Plant Physiol., 93:1203-1211 (1990), andthe tobacco RD2 promoter.

iii. Maturing Endosperm Promoters

In some embodiments, promoters that drive transcription in maturingendosperm can be useful. Transcription from a maturing endospermpromoter typically begins after fertilization and occurs primarily inendosperm tissue during seed development and is typically highest duringthe cellularization phase. Most suitable are promoters that are activepredominantly in maturing endosperm, although promoters that are alsoactive in other tissues can sometimes be used. Non-limiting examples ofmaturing endosperm promoters that can be included in the nucleic acidconstructs provided herein include the napin promoter, the Arcelin-5promoter, the phaseolin promoter (Bustos et al., Plant Cell,1(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs etal., Plant Cell, 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,Plant Mol. Biol., 22(2):255-267 (1993)), the stearoyl-ACP desaturasepromoter (Slocombe et al., Plant Physiol., 104(4):167-176 (1994)), thesoybean α′ subunit of β-conglycinin promoter (Chen et al., Proc. Natl.Acad. Sci. USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al.,Plant Mol. Biol., 34(3):549-555 (1997)), and zein promoters, such as the15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kDzein promoter and 27 kD zein promoter. Also suitable are the Osgt-1promoter from the rice glutelin-1 gene (Zheng et al., Mol. Cell Biol.,13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordeinpromoter. Other maturing endosperm promoters include the YP0092, PT0676,and PT0708 promoters.

iv. Ovary Tissue Promoters

Promoters that are active in ovary tissues such as the ovule wall andmesocarp can also be useful, e.g., a polygalacturonidase promoter, thebanana TRX promoter, the melon actin promoter, YP0396, and PT0623.Examples of promoters that are active primarily in ovules includeYP0007, YP0111, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115,YP0119, YP0120, and YP0374.

v. Embryo Sac/Early Endosperm Promoters

To achieve expression in embryo sac/early endosperm, regulatory regionscan be used that are active in polar nuclei and/or the central cell, orin precursors to polar nuclei, but not in egg cells or precursors to eggcells. Most suitable are promoters that drive expression only orpredominantly in polar nuclei or precursors thereto and/or the centralcell. A pattern of transcription that extends from polar nuclei intoearly endosperm development can also be found with embryo sac/earlyendosperm-preferential promoters, although transcription typicallydecreases significantly in later endosperm development during and afterthe cellularization phase. Expression in the zygote or developing embryotypically is not present with embryo sac/early endosperm promoters.

Promoters that may be suitable include those derived from the followinggenes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsisatmycl (see, Urao (1996) Plant Mol. Biol., 32:571-57; Conceicao (1994)Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); ArabidopsisMEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No.6,906,244). Other promoters that may be suitable include those derivedfrom the following genes: maize MAC1 (see, Sheridan (1996) Genetics,142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) PlantMol. Biol., 22:10131-1038). Other promoters include the followingArabidopsis promoters: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119,YP0137, DME, YP0285, and YP0212. Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

vi. Embryo Promoters

Regulatory regions that preferentially drive transcription in zygoticcells following fertilization can provide embryo-preferentialexpression. Most suitable are promoters that preferentially drivetranscription in early stage embryos prior to the heart stage, butexpression in late stage and maturing embryos is also suitable.Embryo-preferential promoters include the barley lipid transfer protein(Ltp1) promoter (Plant Cell Rep (2001) 20:647-654), YP0097, YP0107,YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, andPT0740.

vii. Photosynthetic Tissue Promoters

Promoters active in photosynthetic tissue confer transcription in greentissues such as leaves and stems. Most suitable are promoters that driveexpression only or predominantly in such tissues. Examples of suchpromoters include the ribulose-1,5-bisphosphate carboxylase (RbcS)promoters such as the RbcS promoter from eastern larch (Larix laricina),the pine cab6 promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778(1994)), the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol.,15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et al.,Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from rice (Luanet al., Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphatedikinase (PPDK) promoter from corn (Matsuoka et al., Proc. Natl. Acad.Sci. USA, 90:9586-9590 (1993)), the tobacco Lhcb1*2 promoter (Cerdan etal., Plant Mol. Biol., 33:245-255 (1997)), the Arabidopsis thaliana SUC2sucrose-H+ symporter promoter (Truernit et al., Planta, 196:564-570(1995)), and thylakoid membrane protein promoters from spinach (psaD,psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissuepromoters include PT0535, PT0668, PT0886, YP0144, YP0380 and PT0585.

viii. Vascular Tissue Promoters

Examples of promoters that have high or preferential activity invascular bundles include YP0087, YP0093, YP0108, YP0022, and YP0080.Other vascular tissue-preferential promoters include the glycine-richcell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell,3(10):1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV)promoter (Medberry et al., Plant Cell, 4(2):185-192 (1992)), and therice tungro bacilliform virus (RTBV) promoter (Dai et al., Proc. Natl.Acad. Sci. USA, 101(2):687-692 (2004)).

ix. Inducible Promoters

Inducible promoters confer transcription in response to external stimulisuch as chemical agents or environmental stimuli. For example, induciblepromoters can confer transcription in response to hormones such asgiberellic acid or ethylene, or in response to light or drought.Examples of drought-inducible promoters include YP0380, PT0848, YP0381,YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384,PT0688, YP0286, YP0377, PD1367, and PD0901. Examples ofnitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886.Examples of shade-inducible promoters include PR0924 and PT0678. Anexample of a promoter induced by salt is rd29A (Kasuga et al. (1999)Nature Biotech 17: 287-291).

x. Basal Promoters

A basal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element that may be locatedbetween about 15 and about 35 nucleotides upstream from the site oftranscription initiation. Basal promoters also may include a “CCAAT box”element (typically the sequence CCAAT) and/or a GGGCG sequence, whichcan be located between about 40 and about 200 nucleotides, typicallyabout 60 to about 120 nucleotides, upstream from the transcription startsite.

xi. Stem Promoters

A stem promoter may be specific to one or more stem tissues or specificto stem and other plant parts. Stem promoters may have high orpreferential activity in, for example, epidermis and cortex, vascularcambium, procambium, or xylem. Examples of stem promoters include YP0018which is disclosed in US20060015970 and CryIA(b) and CryIA(c) (Braga etal. 2003, Journal of New Seeds 5:209-221).

xii. Other Promoters

Other classes of promoters include, but are not limited to,shoot-preferential, callus-preferential, trichome cell-preferential,guard cell-preferential such as PT0678, tuber-preferential, parenchymacell-preferential, and senescence-preferential promoters. Promotersdesignated YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, andYP0096, as described in the above-referenced patent applications, mayalso be useful.

xiii. Other Regulatory Regions

A 5′ untranslated region (UTR) can be included in nucleic acidconstructs described herein. A 5′ UTR is transcribed, but is nottranslated, and lies between the start site of the transcript and thetranslation initiation codon and may include the +1 nucleotide. A 3′ UTRcan be positioned between the translation termination codon and the endof the transcript. UTRs can have particular functions such as increasingmRNA stability or attenuating translation. Examples of 3′ UTRs include,but are not limited to, polyadenylation signals and transcriptiontermination sequences, e.g., a nopaline synthase termination sequence.

It will be understood that more than one regulatory region may bepresent in a recombinant polynucleotide, e.g., introns, enhancers,upstream activation regions, transcription terminators, and inducibleelements. Thus, for example, more than one regulatory region can beoperably linked to the sequence of a polynucleotide encoding a coldtolerance-modulating polypeptide.

Regulatory regions, such as promoters for endogenous genes, can beobtained by chemical synthesis or by subcloning from a genomic DNA thatincludes such a regulatory region. A nucleic acid comprising such aregulatory region can also include flanking sequences that containrestriction enzyme sites that facilitate subsequent manipulation.

IV. Transgenic Plants and Plant Cells A. Transformation

The invention also features transgenic plant cells and plants comprisingat least one recombinant nucleic acid construct described herein. Aplant or plant cell can be transformed by having a construct integratedinto its genome, i.e., can be stably transformed. Stably transformedcells typically retain the introduced nucleic acid with each celldivision. A plant or plant cell can also be transiently transformed suchthat the construct is not integrated into its genome. Transientlytransformed cells typically lose all or some portion of the introducednucleic acid construct with each cell division such that the introducednucleic acid cannot be detected in daughter cells after a sufficientnumber of cell divisions. Both transiently transformed and stablytransformed transgenic plants and plant cells can be useful in themethods described herein.

Transgenic plant cells used in methods described herein can constitutepart or all of a whole plant. Such plants can be grown in a mannersuitable for the species under consideration, either in a growthchamber, a greenhouse, or in a field. Transgenic plants can be bred asdesired for a particular purpose, e.g., to introduce a recombinantnucleic acid into other lines, to transfer a recombinant nucleic acid toother species, or for further selection of other desirable traits.Alternatively, transgenic plants can be propagated vegetatively forthose species amenable to such techniques. As used herein, a transgenicplant also refers to progeny of an initial transgenic plant provided, aslong as the progeny inherits the transgene. Seeds produced by atransgenic plant can be grown and then selfed (or outcrossed and selfed)to obtain seeds homozygous for the nucleic acid construct.

Transgenic plants can be grown in suspension culture, or tissue or organculture. For the purposes of this invention, solid and/or liquid tissueculture techniques can be used. When using solid medium, transgenicplant cells can be placed directly onto the medium or can be placed ontoa filter that is then placed in contact with the medium. When usingliquid medium, transgenic plant cells can be placed onto a flotationdevice, e.g., a porous membrane that contacts the liquid medium. A solidmedium can be, for example, Murashige and Skoog (MS) medium containingagar and a suitable concentration of an auxin, e.g.,2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration ofa cytokinin, e.g., kinetin.

When transiently transformed plant cells are used, a reporter sequenceencoding a reporter polypeptide having a reporter activity can beincluded in the transformation procedure and an assay for reporteractivity or expression can be performed at a suitable time aftertransformation. A suitable time for conducting the assay typically isabout 1-21 days after transformation, e.g., about 1-14 days, about 1-7days, or about 1-3 days. The use of transient assays is particularlyconvenient for rapid analysis in different species, or to confirmexpression of a heterologous cold tolerance-modulating polypeptide whoseexpression has not previously been confirmed in particular recipientcells.

Techniques for introducing nucleic acids into monocotyledonous anddicotyledonous plants are known in the art, and include, withoutlimitation, Agrobacterium-mediated transformation, viral vector-mediatedtransformation, electroporation and particle gun transformation, e.g.,U.S. Pat. Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cellor cultured tissue is used as the recipient tissue for transformation,plants can be regenerated from transformed cultures if desired, bytechniques known to those skilled in the art.

B. Screening/Selection

A population of transgenic plants can be screened and/or selected forthose members of the population that have a trait or phenotype conferredby expression of the transgene. For example, a population of progeny ofa single transformation event can be screened for those plants having adesired level of expression of a cold tolerance-modulating polypeptideor nucleic acid. Physical and biochemical methods can be used toidentify expression levels. These include Southern analysis or PCRamplification for detection of a polynucleotide; Northern blots, 51RNAse protection, primer-extension, or RT-PCR amplification fordetecting RNA transcripts; enzymatic assays for detecting enzyme orribozyme activity of polypeptides and polynucleotides; and protein gelelectrophoresis, Western blots, immunoprecipitation, and enzyme-linkedimmunoassays to detect polypeptides. Other techniques such as in situhybridization, enzyme staining, and immunostaining also can be used todetect the presence or expression of polypeptides and/orpolynucleotides. Methods for performing all of the referenced techniquesare known. As an alternative, a population of plants comprisingindependent transformation events can be screened for those plantshaving a desired trait, such as a modulated level of cold tolerance.Selection and/or screening can be carried out over one or moregenerations, and/or in more than one geographic location. In some cases,transgenic plants can be grown and selected under conditions whichinduce a desired phenotype or are otherwise necessary to produce adesired phenotype in a transgenic plant. In addition, selection and/orscreening can be applied during a particular developmental stage inwhich the phenotype is expected to be exhibited by the plant. Selectionand/or screening can be carried out to choose those transgenic plantshaving a statistically significant difference in a cold tolerance levelrelative to a control plant that lacks the transgene. Selected orscreened transgenic plants have an altered phenotype as compared to acorresponding control plant, as described in the “Transgenic PlantPhenotypes” section herein.

C. Plant Species

The polynucleotides and vectors described herein can be used totransform a number of monocotyledonous and dicotyledonous plants andplant cell systems, including species from one of the followingfamilies: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae,Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae,Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae,Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae,Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae,Theaceae, or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

Suitable species include Panicum spp. or hybrids thereof, Sorghum spp.or hybrids thereof, sudangrass, Miscanthus spp. or hybrids thereof,Saccharum spp. or hybrids thereof, Erianthus spp., Populus spp.,Andropogon gerardii (big bluestem), Pennisetum purpureum (elephantgrass) or hybrids thereof (e.g., Pennisetum purpureum×Pennisetumtyphoidum), Phalaris arundinacea (reed canarygrass), Cynodon dactylon(bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata(prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giantreed) or hybrids thereof, Secale cereale (rye), Salix spp. (willow),Eucalyptus spp. (eucalyptus), Triticosecale (Triticum—wheat X rye),Tripsicum dactyloides (Eastern gammagrass), Leymus cinereus (basinwildrye), Leymus condensatus (giant wildrye), and bamboo.

In some embodiments, a suitable species can be a wild, weedy, orcultivated sorghum species such as, but not limited to, Sorghum almum,Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum bicolor(such as bicolor, guinea, caudatum, kafir, and durra), Sorghumbrachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum,Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande,Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghumlaxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghummatarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum,Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghumstipoideum, Sorghum sudanensese, Sorghum timorense, Sorghumtrichocladum, Sorghum versicolor, Sorghum virgatum, Sorghum vulgare, orhybrids such as Sorghum×almum, Sorghum×sudangrass or Sorghum×drummondii.

Suitable species also include Helianthus annuus (sunflower), Carthamustinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis(castor), Elaeis guineensis (palm), Linum usitatissimum (flax), andBrassica juncea.

Suitable species also include Beta vulgaris (sugarbeet), and Manihotesculenta (cassava).

Suitable species also include Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), andSolanum melongena (eggplant).

Suitable species also include Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Colchicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (=Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, and Tanacetum parthenium.

Suitable species also include Parthenium argentatum (guayule), Heveaspp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixaorellana, and Alstroemeria spp.

Suitable species also include Rosa spp. (rose), Dianthus caryophyllus(carnation), Petunia spp. (petunia) and Poinsettia pulcherrima(poinsettia).

Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus(lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populustremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp.(maple, Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp.(ryegrass) and Phleum pratense (timothy).

Thus, the methods and compositions can be used over a broad range ofplant species, including species from the dicot genera Brassica,Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium,Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum,Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale,Sorghum, Triticosecale, Triticum, and Zea. In some embodiments, a plantis a member of the species Panicum virgatum (switchgrass), Sorghumbicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus),Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays(corn), Glycine max (soybean), Brassica napus (canola), Triticumaestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice),Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris(sugarbeet), or Pennisetum glaucum (pearl millet).

In certain embodiments, the polynucleotides and vectors described hereincan be used to transform a number of monocotyledonous and dicotyledonousplants and plant cell systems, wherein such plants are hybrids ofdifferent species or varieties of a specific species (e.g., Saccharumsp.×Miscanthus sp., Panicum virgatum×Panicum amarum, Panicumvirgatum×Panicum amarulum, and Pennisetum purpureum×Pennisetumtyphoidum).

D. Transgenic Plant Phenotypes

In some embodiments, a plant in which expression of a coldtolerance-modulating polypeptide is modulated can have increased levelsof cold tolerance and/or biomass in vegetative tissues. Cold tolerancecan be measured by means well know to those of skill in the art,including, but not limited to, seedling survival, decreasedphotosynthesis and membrane damage (measured by electrolyte leakage),seedling area, yield, and or biomass. For example, a coldtolerance-modulating polypeptide or nucleic acid described herein can beexpressed in a transgenic plant, resulting in increased levels of coldtolerance and/or biomass. The cold tolerance level can be increased byat least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100or more than 100 percent, as compared to the cold tolerance level in acorresponding control plant that does not express the transgene. In someembodiments, a plant in which expression of a cold tolerance-modulatingpolypeptide or polynucleotide is modulated can have increased levels ofbiomass. The biomass level can be increased by at least 2 percent, e.g.,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or morethan 100 percent, as compared to the biomass level in a correspondingcontrol plant that does not express the transgene. In some embodiments,differences can be measured for a plant in which expression of a coldtolerance-modulating polypeptide is modulated can be exposed to cold forone or more periods of time that may vary depending on climaticconditions. For example, for periods of about ½ hour, 1 hour, 3 hours, 6hours, 12 hours, 1 day, 3 days, 5 days, 10 days, 1 month, 3 months, 6months, 12 months, or the entire lifespan of such a plant.

Increases in cold tolerance in such plants can provide improvednutritional quantity and content in geographic locales where coldaffects plants. Increases in cold tolerance in such plants can be usefulin situations where plant parts such as, but not limited to, seeds,tubers, stems, leaves or roots are harvested for human or animalconsumption.

Decrease in cold tolerance in such plants can be useful for species orvarieties of plants that benefit from cold exposure. For example, coldsensitive plants might be able to undergo vernalization more easily.Decreases in cold tolerance in such plants can be useful in situationswhere plant parts such as, but not limited to, seeds, tubers, stems,leaves or roots are harvested for human or animal consumption.

Typically, a difference in the level of cold tolerance in a transgenicplant or cell relative to a control plant or cell is consideredstatistically significant at p≦0.05 with an appropriate parametric ornon-parametric statistic, e.g., Chi-square test, Student's t-test,Mann-Whitney test, or F-test. In some embodiments, a difference in thelevel of cold tolerance is statistically significant at p<0.01, p<0.005,or p<0.001. A statistically significant difference in, for example, thelevel of cold tolerance in a transgenic plant compared to the amount incells of a control plant indicates that the recombinant nucleic acidpresent in the transgenic plant results in altered cold tolerancelevels.

The phenotype of a transgenic plant is evaluated relative to a controlplant. A plant is said “not to express” a polypeptide when the plantexhibits less than 10%, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount of polypeptide or mRNAencoding the polypeptide exhibited by the plant of interest. Expressioncan be evaluated using methods including, for example, RT-PCR, Northernblots, S1 RNAse protection, primer extensions, Western blots, proteingel electrophoresis, immunoprecipitation, enzyme-linked immunoassays,chip assays, and mass spectrometry. It should be noted that if apolypeptide is expressed under the control of a tissue-preferential orbroadly expressing promoter, expression can be evaluated in the entireplant or in a selected tissue. Similarly, if a polypeptide is expressedat a particular time, e.g., at a particular time in development or uponinduction, expression can be evaluated selectively at a desired timeperiod.

V. Plant Breeding

Genetic polymorphisms are discrete allelic sequence differences in apopulation. Typically, an allele that is present at 1% or greater isconsidered to be a genetic polymorphism. The discovery that polypeptidesdisclosed herein can modulate cold tolerance content is useful in plantbreeding, because genetic polymorphisms exhibiting a degree of linkagewith loci for such polypeptides are more likely to be correlated withvariation in a cold tolerance trait. For example, genetic polymorphismslinked to the loci for such polypeptides are more likely to be useful inmarker-assisted breeding programs to create lines having a desiredmodulation in the cold tolerance trait.

Thus, one aspect of the invention includes methods of identifyingwhether one or more genetic polymorphisms are associated with variationin a cold tolerance trait. Such methods involve determining whethergenetic polymorphisms in a given population exhibit linkage with thelocus for one of the polypeptides depicted in FIGS. 1 to 5 and/orfunctional homologs thereof, such as, but not limited to thoseidentified in the Sequence Listing of this application. The correlationis measured between variation in the cold tolerance trait in plants ofthe population and the presence of the genetic polymorphism(s) in plantsof the population, thereby identifying whether or not the geneticpolymorphism(s) are associated with variation for the trait. If thepresence of a particular allele is statistically significantlycorrelated with a desired modulation in the cold tolerance trait, theallele is associated with variation for the trait and is useful as amarker for the trait. If, on the other hand, the presence of aparticular allele is not significantly correlated with the desiredmodulation, the allele is not associated with variation for the traitand is not useful as a marker.

Such methods are applicable to populations containing the naturallyoccurring endogenous polypeptide rather than an exogenous nucleic acidencoding the polypeptide, i.e., populations that are not transgenic forthe exogenous nucleic acid. It will be appreciated, however, thatpopulations suitable for use in the methods may contain a transgene foranother, different trait, e.g., herbicide resistance.

Genetic polymorphisms that are useful in such methods include simplesequence repeats (SSRs, or microsatellites), rapid amplification ofpolymorphic DNA (RAPDs), single nucleotide polymorphisms (SNPs),amplified fragment length polymorphisms (AFLPs) and restriction fragmentlength polymorphisms (RFLPs). SSR polymorphisms can be identified, forexample, by making sequence specific probes and amplifying template DNAfrom individuals in the population of interest by PCR. If the probesflank an SSR in the population, PCR products of different sizes will beproduced. See, e.g., U.S. Pat. No. 5,766,847. Alternatively, SSRpolymorphisms can be identified by using PCR product(s) as a probeagainst Southern blots from different individuals in the population.See, U. H. Refseth et al., (1997) Electrophoresis 18: 1519. Theidentification of RFLPs is discussed, for example, in Alonso-Blanco etal. (Methods in Molecular Biology, vol. 82, “Arabidopsis Protocols”, pp.137-146, J. M. Martinez-Zapater and J. Salinas, eds., c. 1998 by HumanaPress, Totowa, N.J.); Burr (“Mapping Genes with Recombinant Inbreds”,pp. 249-254, in Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c.1994 by Springer-Verlag New York, Inc.: New York, N.Y., USA; BerlinGermany; Burr et al. Genetics (1998) 118: 519; and Gardiner, J. et al.,(1993) Genetics 134: 917). The identification of AFLPs is discussed, forexample, in EP 0 534 858 and U.S. Pat. No. 5,878,215.

In some embodiments, the methods are directed to breeding a plant line.Such methods use genetic polymorphisms identified as described above ina marker assisted breeding program to facilitate the development oflines that have a desired alteration in the cold tolerance trait. Once asuitable genetic polymorphism is identified as being associated withvariation for the trait, one or more individual plants are identifiedthat possess the polymorphic allele correlated with the desiredvariation. Those plants are then used in a breeding program to combinethe polymorphic allele with a plurality of other alleles at other locithat are correlated with the desired variation. Techniques suitable foruse in a plant breeding program are known in the art and include,without limitation, backcrossing, mass selection, pedigree breeding,bulk selection, crossing to another population and recurrent selection.These techniques can be used alone or in combination with one or moreother techniques in a breeding program. Thus, each identified plants isselfed or crossed a different plant to produce seed which is thengerminated to form progeny plants. At least one such progeny plant isthen selfed or crossed with a different plant to form a subsequentprogeny generation. The breeding program can repeat the steps of selfingor outcrossing for an additional 0 to 5 generations as appropriate inorder to achieve the desired uniformity and stability in the resultingplant line, which retains the polymorphic allele. In most breedingprograms, analysis for the particular polymorphic allele will be carriedout in each generation, although analysis can be carried out inalternate generations if desired.

In some cases, selection for other useful traits is also carried out,e.g., selection for fungal resistance or bacterial resistance. Selectionfor such other traits can be carried out before, during or afteridentification of individual plants that possess the desired polymorphicallele.

VI. Articles of Manufacture

Transgenic plants provided herein have various uses in the agriculturaland energy production industries. For example, transgenic plantsdescribed herein can be used to make animal feed and food products. Suchplants, however, are often particularly useful as a feedstock for energyproduction.

Transgenic plants described herein often produce higher yields of grainand/or biomass per hectare, relative to control plants that lack theexogenous nucleic acid. In some embodiments, such transgenic plantsprovide equivalent or even increased yields of grain and/or biomass perhectare relative to control plants when grown under conditions ofreduced inputs such as fertilizer and/or water. Thus, such transgenicplants can be used to provide yield stability at a lower input costand/or under environmentally stressful conditions such as drought. Insome embodiments, plants described herein have a composition thatpermits more efficient processing into free sugars, and subsequentlyethanol, for energy production. In some embodiments, such plants providehigher yields of ethanol, butanol, other biofuel molecules, and/orsugar-derived co-products per kilogram of plant material, relative tocontrol plants. Such processing efficiencies are believed to be derivedfrom the chemical composition of the plant material. By providing higheryields at an equivalent or even decreased cost of production relative tocontrol plants that do not have increased levels of cold tolerance, thetransgenic plants described herein improve profitability for farmers andprocessors as well as decrease costs to consumers.

Seeds from transgenic plants described herein can be conditioned andbagged in packaging material by means known in the art to form anarticle of manufacture. Packaging material such as paper and cloth arewell known in the art. A package of seed can have a label, e.g., a tagor label secured to the packaging material, a label printed on thepackaging material, or a label inserted within the package, thatdescribes the nature of the seeds therein.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

VII. Examples Example 1 Transgenic Arabidopsis Plants

The following symbols are used in the Examples with respect toArabidopsis transformation: T1: first generation transformant; T2:second generation, progeny of self-pollinated T1 plants; T3: thirdgeneration, progeny of self-pollinated T2 plants; T4: fourth generation,progeny of self-pollinated T3 plants. Independent transformations arereferred to as events.

The following is a list of nucleic acids that were isolated fromArabidopsis thaliana plants, Clone 2273, Clone 924103, and Clone 13209.The nucleic acids designated Clone 6639 and Clone 924103 were isolatedfrom the species Triticum aestivum.

Each isolated nucleic acid described above was cloned into a Ti plasmidvector containing a phosphinothricin acetyltransferase gene whichconfers Finale™ resistance to transformed plants. A Ti plasmid vectoruseful for these constructs is CRS 338. Unless otherwise indicated, eachCeres Clone and/or Seedline derived from a Clone is in the senseorientation relative to either the 35S promoter in a Ti plasmid.Wild-type Arabidopsis thaliana ecotype Wassilewskija (Ws) plants weretransformed separately with each construct. The transformations wereperformed essentially as described in Bechtold et al., C.R. Acad. Sci.Paris, 316:1194-1199 (1993).

Wild-type Arabidopsis Wassilewskija (Ws) plants were transformed with aTi plasmid containing Clone 924103 in the sense orientation relative tothe 326F promoter. The Ti plasmid vector used for this construct,CRS814, contains the Ceres-constructed, plant selectable marker genephosphinothricin acetyltransferase (PAT) which confers herbicideresistance to transformed plants.

Wild-type Arabidopsis Wassilewskija (Ws) plants were transformed with aTi plasmid containing clone 2273 in the sense orientation relative tothe 32449 promoter.

Wild-type Arabidopsis Wassilewskija (Ws) plants were transformed with aTi plasmid containing clone 13209 in the sense orientation relative tothe 32449 promoter. The Ti plasmid vector used for this construct,CRS311, contains the Ceres-constructed, plant selectable marker genephosphinothricin acetyltransferase (PAT) which confers herbicideresistance to transformed plants

Transgenic Arabidopsis lines containing Clone 2273, Clone 6639, Clone924103, or Clone 13209 were designated ME00327, ME04315, ME17294, orME00572 respectively. The presence of each vector containing a nucleicacid described above in the respective transgenic Arabidopsis linetransformed with the vector was confirmed by Finale™ resistance, PCRamplification from green leaf tissue extract, and/or sequencing of PCRproducts. As controls, wild-type Arabidopsis ecotype Ws plants weretransformed with the empty vector SR0059.

Example 2 Screening for Cold Tolerance in Transgenic Plants

How plants respond to stress in the environment dictates their abilityto survive and reproduce. There are probably many mechanisms by whichplants regulate the temperatures under which they will germinate (Lu andHills, 2003). A number of polynucleotides that result in stresstolerance when over-expressed have been identified in model species suchas Arabidopsis.

Over-expression of these polynucleotides could be useful for increasinglow temperature, chilling or cold tolerance in crops. Assays describedhere focus on low temperature, chilling or cold tolerance in seedlings.The ability to germinate and grow under low temperature, chilling orcold, and wet conditions would allow a longer growing season andmitigate damage caused by unexpected low temperature, chilling or coldperiods. If this trait is recapitulated in crops overexpressing thesepolynucleotides, the result could be very valuable in agriculture inmany crops and environments and make a significant contribution tosustainable farming. Furthermore, low temperature, chilling or coldtolerance may be modulated by expressing these polynucleotides under thecontrol of a low temperature, chilling or cold inducible promoter.

1. Cold Growth Superpool Screen

Plates of solidified agar MS medium are prepared for the screen asfollows. One liter of medium is prepared by mixing 2.15 g of MS basalsalt mixture (from Phytotech M524) and 7 g of agar (from EM Science,1.01614.1000) in water, and adjusting the pH to 5.7 with a 10N KOHsolution. After autoclaving, 45 ml of media are transferred understerile conditions per 100 mm square×15 mm deep plate.

Individual superpool and control seeds are sterilized in a 30% bleachsolution for 5 minutes. Seeds are then rinsed repeatedly with sterilewater to eliminate all bleach solution. Seeds are plated using a COPAS™robot (Union Biometrica, Holliston, Mass.) at a density of 72 seeds perplate. The plates are wrapped with vent tape and transferred to a dark4° C. refrigerator for 3 days to promote uniform germination. The platesare then placed horizontally in a Conviron growth chamber set at 22° C.,16:8 hour light:dark cycle, 70% humidity with fluorescent lamps emittinga light intensity of ˜100 μEinsteins. Normal growth is allowed to occurfor 3-5 days. At end of 3-5 days of growth, images of the plates arescanned using an Epson perfection 4870 scanner. Then, cold-growthtreatment is applied for 1-3 weeks. Accordingly, plates are transferredin a horizontal position to an 8° C. Conviron chamber under constantlight at ˜100 μEinsteins. After a defined number of days of cold-growthtreatment, for example 7 or 14, the plates are scanned again. TheWinRhizo software program (Regent Instruments Inc., Canada) is used todetermine the area for each seedling from the scanned images.

Individual seedlings that perform better in the cold growth screen areidentified by visual inspection for those showing obvious morphologicaldifferences and by statistical analysis of the seedling area data. DNAfrom these candidate seedlings is extracted and the transgene amplifiedusing PCR. The PCR product is sequenced to determine the identity of thetransgene and consequently the ME line from which the candidate isderived.

2. Cold Growth Assay

The cold growth assay is used to validate candidate misexpression (ME)lines obtained from screens for enhanced growth under cold conditions.This procedure allows a high-throughput methodology for assessingtransgenic Arabidopsis candidates that have germinated at normaltemperature (22° C.) and light (˜100 to 200 μEinsteins) in a walk-ingrowth chamber on agar solidified MS medium before transfer to coldtemperature. It relies on the ability to discriminate between seedlingsthat have become significantly larger during cold growth than controlsby imaging the seedlings when they are transferred to the cold and thenperiodically thereafter under cold growth conditions.

Plate preparation for the cold growth assay and the growth conditionsare the same as those described for the cold growth screen as describedabove. Seeds from independent transformation events for each ME line arebleach sterilized and then plated at a density of 40 seeds per plate (30seeds from the event and 10 wild-type control seeds). After cold-growthtreatment, the seedlings are then FINALE®-treated to identify the plantscarrying the ME vector.

Cold growth is characterized by statistical analysis as follows. Thecontrol population is the internal non-transgenic segregants for thatparticular event. When there are not enough internal non-transgenicsegregants for an event, a pool of all non-transgenic segregants fromall events associated with that ME line is used (i.e. whennon-transgenics are less than five for the event or the event appears tobe homozygous). Pooling is only done for events associated with the sameME line and within an experiment (an experiment is the set of plateswith a common sow date). Thus in the final analysis, the pooled controlpopulation may be different for generations T₂ and T₃.

The WinRhizo software program (Regent Instruments Inc., Canada) is usedto determine the area for each seedling. The change in area iscalculated for a defined number of days of treatment. A one-tailedt-test is used to compare change in area and the mean size of thetransgenic seedlings within an event to the internal non-transgenicsegregants. Significance is assessed at an α-value of 0.05.

3. Cold Flux Assay

The cold flux growth assay is used to validate candidate misexpression(ME) lines obtained from screens for enhanced growth under fluctuatingcold conditions. This procedure allows a high-throughput methodology forassessing transgenic Arabidopsis candidates that have germinated atnormal temperature (22° C.) and light (˜100 to 200 μEinsteins) in awalk-in growth chamber on agar solidified MS medium before transfer tocold temperature. It relies on the ability to discriminate betweenseedlings that have become significantly larger during growth underfluctuating cold conditions than controls by imaging the seedlings whenthey are transferred to the cold and then periodically thereafter undercold growth conditions.

Plate preparation for the cold growth assay and the growth conditionsare the same as those described for the cold growth screen as describedabove. Seeds from independent transformation events for each ME line arebleach sterilized and then plated at a density of 61 seeds per plate(including both seeds from the event and wild-type control seeds). Aftercold flux-growth treatment, the seedlings are then FINALE®-treated toidentify the plants carrying the ME vector.

Normal growth is allowed to occur for 3-5 days. At end of 3-5 days ofgrowth, images of the plates are scanned using an Epson perfection 4870scanner. After 3-5 days growth in normal conditions, the plates aretransferred in a horizontal position to an 8° C. Conviron under constantlight at ˜100 μEinsteins. All transfers take place in the morning.Growth is allowed at 8° C. for 3-4 days. After 3-4 days growth at 8° C.,plates are transferred to 1° C. Percival under constant light at ˜70μEinsteins. Growth is allowed at 1° C. for 3-4 days. 8° C./1° C. cyclingis repeated for a total of 14 days. The plates are imaged using CFimager and Winrhizo scanner. Individual seedlings are selected which aresignificantly larger and/or exhibit increased photosynthetic efficiency(Fv/Fm). Plates are visually observed as well. DNA from these candidateseedlings is extracted and the transgene amplified using PCR. The PCRproduct is sequenced to determine the identity of the transgene andconsequently the ME line from which the candidate is derived. Theseseedlings are then grown for progeny seed.

Cold flux growth is characterized by statistical analysis as follows.The control population is the internal non-transgenic segregants forthat particular event. When there are not enough internal non-transgenicsegregants for an event, a pool of all non-transgenic segregants fromall events associated with that ME line is used (i.e. whennon-transgenics are less than five for the event or the event appears tobe homozygous). Pooling is only done for events associated with the sameME line and within an experiment (an experiment is the set of plateswith a common sow date). Thus in the final analysis, the pooled controlpopulation may be different for generations T2 and T3.

The WinRhizo software program (Regent Instruments Inc., Canada) is usedto determine the area for each seedling. The change in area iscalculated for a defined number of days of treatment. A one-tailedt-test is used to compare change in area and the mean size of thetransgenic seedlings within an event to the internal non-transgenicsegregants. Significance is assessed at an α-value of 0.05.

Example 3 Results for ME00327 Events (SEQ ID NO:2)

Ectopic expression of clone 2273 (from Arabidopsis thaliana) under thecontrol of the 32449 promoter in the ME00327 plants results in largerseedlings after 14 days fluctuation between 8° C. and 1° C.

The seedling area of transgenic plants within a seed line was comparedto that of non-transgenic segregants within the same seed line after 14days of growth at fluctuating temperatures of 8° C. and 1° C. Six eventsof ME00327 were analyzed as described in the cold flux assay (Example2). Events-04 and -06 were significant in at least two generations atp<0.05 using a one-tailed t-test assuming unequal variance (Table 1).The transgenic plants were visibly larger than the controls.

Event-04 segregated 3:1 (R:S) for Finale™ resistance in the T₂generation. Event-06 appears to segregate for two inserts (15:1) in theT₂ generation.

TABLE 1 t-test comparison of seedling area between transgenic seedlingsand control non-transgenic segregants after 14 days fluctuation between8° C. and 1° C. Control Event- Transgenic Non-Transgenics^(a) t-testEvents Gen Avg SE N Avg SE N p-value ME00327-04 -04-T2 0.0383 0.0014 160.0297 0.0011 14 2.58E−05 ME00327-04- -04-T3 0.0490 0.0023 27 0.04100.0019 32 4.48E−03 02 ME00327-04- -04-T3 0.0522 0.0018 32 0.0378 0.002026 9.46E−07 03 ME00327-04- -04-T3 0.0497 0.0020 35 0.0364 0.0022 251.38E−05 04 ME00327-06^(c) -06-T2 0.0314 0.0011 29 0.0272 0.0009 391.98E−03 ME00327-06^(bc) -06-T2 0.0392 0.0012 54 0.0346 0.0008 1948.17E−04 ME00327-06- -06-T3 0.0296 0.0015 41 0.0232 0.0016 17 2.47E−0301 ME00327-06- -06-T3 0.0356 0.0015 42 0.0290 0.0019 15 4.44E−03 03^(a)Transgenic seedlings were compared to internal non-transgenicsegregants within an event unless otherwise indicated. ^(b)These eventswere sown twice. The first time was to identify ME00327 as a Hit. Theywere repeated the second time with the next generation to identifyME00327 as a Lead. ^(c)These events did not segregate non-transgenicseedlings and were compared to pooled non-transgenics for the line.

Plants from Events-04 and -06 which are hemizygous or homozygous forclone 2273 do not show any negative phenotypes under standardconditions. Events-04 and -06 of ME00327 were tested for negativephenotypes compared to the empty vector control SR00559. The resultsshowed no detectable reduction in germination rate, the plants appearedwild-type in all instances, and no statistical differences in days toflowering, rosette area 7 days post-bolting, or fertility (siliquenumber and seed fill).

Example 4 Results for ME04315 Events (SEQ ID NO: 20)

Candidate ME04315 was identified by superpool screen described above inExample 2. Ectopic expression of Clone 6639 under the control of the 35Spromoter in ME04315 plants results in early germination at 8° C.resulting in larger seedlings after 10 days at 8° C.

The seedling area of transgenic plants within a seed line was comparedto that of non-transgenic segregants within the same seed line after 10days of growth at 8° C. Nine events of ME04315 were analyzed asdescribed in the Cold Growth Assay described in Example 2 and showedsignificant tolerance under cold conditions in two generations. ThreeEvents, -02, -03 and -06, were significant in both generations at p<0.05using a one-tailed t-test assuming unequal variance (Table 2). ‘-99’signifies that seeds were pooled from several plants. Events-02 and -06were from the T3 generation because T2 seed was not available.Subsequently, next generation seeds for three of the events (T3 or T4 asneeded) were evaluated under cold germination conditions.

The transgenic plants were visibly larger and lighter in color than thecontrols. Under cold conditions, seedlings typically become darker,presumably due to the accumulation of anthocyanin The lighter colorexhibited by ME04315 seedlings suggests a decrease in this stressresponse. ME04315 plants grown under standard conditions in soil did notappear different in color than controls.

Event-03 segregated 3:1 (R:S) for Finale™ resistance in the T₂generation. Seed collected from individual, hemizygous plants was notavailable for Events-02 and -06. However, the T₃ generation seeds thatwere pooled from several T₂ plants segregated approximately 2:1 in amanner consistent with a single insert for Event-06 (only transgenicplants were pooled). Pooled T₃ generation seeds for Event-02 segregated1:3.

TABLE 2 T-test comparison of seedling area between transgenic seedlingsand control non-transgenic segregants after 10 days at 8° C. ControlNon- Event- Transgenic Transgenics^(a) t-test Events Gen Avg SE N Avg SEN p-value ME04315-02-99^(b) 02-T3 0.0071 0.0013 10 0.0049 0.0007 247.50E−02 ME04315-02-99 02-T3 0.0046 0.0007 16 0.0036 0.0002 50 7.92E−02ME04315-02-99-02^(a) 02-T4 0.0070 0.0002 61 0.0047 0.0002 213 6.66E−16ME04315-02-99-03 02-T4 0.0072 0.0002 45 0.0057 0.0007 3 2.08E−02ME04315-03^(b) 03-T2 0.0026 0.0002 18 0.0017 0.0003 7 0.0070 ME04315-0303-T2 0.0017 0.0001 35 0.0016 0.0001 19 0.2221 ME04315-03-02 03-T30.0078 0.0005 12 0.0045 0.0011 7 6.69E−03 ME04315-03-03 03-T3 0.00630.0003 45 0.0053 0.0004 24 0.0153 ME04315-06-99^(b) 06-T3 0.0034 0.000616 0.0023 0.0003 14 0.0499 ME04315-06-99 06-T3 0.0029 0.0002 42 0.00210.0001 28 9.52E−04 ME04315-06-99-02^(a) 06-T4 0.0072 0.0002 61 0.00470.0002 213 3.33E−16 ^(a)Transgenic seedlings were compared tonon-transgenic segregants within a seed line except for the T₄generation of Events -02 and -06. Since these seed lines werehomozygous, they were compared to pooled non-transgenic segregants fromanother T₄ generation event that was grown in the same flat as the T₄generation of Events -02 and -06. ^(b)These events were sown twice. Thefirst time was to identify ME04315 as a hit. They were repeated thesecond time with two generations to identify ME04315 as a Lead.

Plants from Events-02, -03 and -06 which are hemizygous or homozygousfor Clone 6639 do not show any negative phenotypes under long-dayconditions. The physical appearance of eight of the nine T₁ plants wasidentical to the controls. Event-06 was smaller and had fewer rosetteleaves.

Events-02, -03 and -06 of ME04315 exhibited no statistically significantnegative phenotypes compared to empty vector control SR00559. There wasno detectable reduction in germination rate, the plants appearedwild-type in all instances, and there was no observable or statisticaldifferences between experimentals and controls for days to flowering,rosette area 7 days post-bolting or fertility (silique number and seedfill).

Example 5 Results for ME17294 Events (SEO ID NO:93) 5′ Truncated

Nine events of ME17294 (Clone 924103 from Triticum aestivum) wereanalyzed as described in the cold germination assay (Example 2). In thisstudy, the seedling area (a measure of germination timing and cotyledonexpansion) of transgenic plants within a seed line was compared to thatof non-transgenic segregants within the same seed line, except for theT3 generation of both events. These seed lines were homozygous for thetransgene. For these seed lines, we used pooled non-transgenicsegregants from another T3 generation event of ME17294 that werecollected from plants grown in the same flat as the T3 generation ofEvents-08 and -09.

The two events, -08 and -09, were significant in two generations atp<0.05 using a one-tailed t-test assuming unequal variance (Table 3).The transgenic plants are visibly larger.

Events-08 and -09 segregated 3:1 (R:S) for Finale™ resistance in the T₂generation. No T₁ phenotypes were reported for this line.

TABLE 3 t-test comparison of seedling area between transgenic seedlingsand control non-transgenic segregants after 10 days at 8° C. ControlNon- Event- Transgenic Transgenics^(a) t-test Events Gen Avg SE N Avg SEN p-value ME17294-08^(b) 08-T2 0.0032 0.0003 24 0.0022 0.0002 9 2.57E−03ME17294-08 08-T2 0.0021 0.0001 41 0.0018 0.0001 10 4.52E−03ME17294-08-02^(a) 08-T3 0.0083 0.0003 48 0.0064 0.0002 234 3.05E−08ME17294-08-04 08-T3 0.0075 0.0003 54 0.0064 0.0002 234 9.19E−04ME17294-09^(b) 09-T2 0.0058 0.0003 22 0.0035 0.0002 9 1.02E−06ME17294-09 09-T2 0.0039 0.0002 41 0.0029 0.0002 16 4.59E−04ME17294-09-01^(a) 09-T3 0.0073 0.0003 46 0.0064 0.0002 234 5.53E−03ME17294-09-04^(a) 09-T3 0.0087 0.0003 63 0.0064 0.0002 234 −9.10E−11^(a)Transgenic seedlings were compared to internal non-transgenicsegregants within a seed line except for the T₃ generation of Events-08and -09. Since these seed lines were homozygous, they were compared topooled non-transgenic segregants from another T₃ generation event thatwas grown in the same flat as the T₃ generation of Events -08 and -09.^(b)These events were sown twice. The first time was to identify ME17294as a Hit. They were repeated the second time with two generations toidentify ME17294 as a Lead.

Plants from Events-08 and -09 which are hemizygous or homozygous forclone 924103 do not show any negative phenotypes under standardconditions. Events-08 and -09 of ME17294 exhibited no statisticallysignificant negative phenotypes compared to empty vector controlSR00559. There was no detectable reduction in germination rate, theplants appeared wild-type in all instances, and there were nostatistical differences between experimentals and controls for days toflowering, rosette area 7 days post-bolting, or fertility (siliquenumber and seed fill).

Example 6 Results for ME00572 Events (SEO ID NO:111) tasiRNA

Clone 13209, in ME00572 plants, is a trans-acting small interfering RNA(tasiRNA) that interacts with ARFs (Auxin Response Factors). A megapoolcontaining superpools 9-12 was screened for seedlings that grew morevigorously than controls after transfer to fluctuating cold conditionsaccording to Example 2. Seven candidates were chosen from this megapool.ME00572 was represented two times in this set.

Four events of ME00572 showed significant tolerance under coldfluctuating conditions in at least two generations. The seedling area oftransgenic plants within a seed line was compared to that ofnon-transgenic segregants within the same seed line after 14 days ofgrowth at fluctuating temperatures of 8° C. and 1° C. Five events ofME00572 were analyzed as described in the Cold Flux Assay described inExample 2. Events-01, -03, -04 and -05 were significant in at least twogenerations at p<0.05 using a one-tailed t-test assuming unequalvariance (Table 4). The transgenic plants were visibly larger than thecontrols.

Events-01 and -05 segregated 3:1 (R:S) for Finale™ resistance in the T2generation. Event-04 segregated 3:1 in the T3 generation. Event-03segregated 1:1 in the T2 generation.

TABLE 4 t-test comparison of seedling area between transgenic seedlingsand control non-transgenic segregants after 14 days fluctuation between8° C. and 1° C. Control Event- Transgenic Non-Transgenics^(a) t-testEvents Gen Avg SE N Avg SE N p-value ME00572-01 -01-T2 0.0338 0.0293 260.0233 0.0010 4 0.155617 ME00572-01^(b) -01-T2 0.0541 0.0021 45 0.04420.0030 13 4.92E−03 ME00572-01- -01-T3 0.0347 0.0017 40 0.0246 0.0014 141.53E−05 01 ME00572-01- -01-T3 0.0331 0.0013 59 0.0267 0.0005 2942.35E−06 02^(c) ME00572-01- -01-T3 0.0360 0.0010 59 0.0267 0.0005 294−9.39E−11 03^(c) ME00572-01- -01-T3 0.0329 0.0013 46 0.0233 0.0018 143.19E−05 04 ME00572-03 -03-T2 0.0312 0.0016 11 0.0223 0.0013 19 9.71E−05ME00572-03^(b) -03-T2 0.0383 0.0024 24 0.0341 0.0023 25 1.04E−01ME00572-03- -03-T3 0.0328 0.0014 30 0.0236 0.0008 28 1.42E−07 01ME00572-03- -03-T3 0.0311 0.0010 26 0.0261 0.0015 33 3.81E−03 02ME00572-03- -03-T3 0.0352 0.0011 28 0.0252 0.0009 32 1.39E−09 03ME00572-03- -03-T3 0.0331 0.0019 23 0.0260 0.0014 35 1.97E−03 04ME00572-04- -04-T3 0.0235 0.0009 23 0.0158 0.0020 7 6.98E−04 99ME00572-04- -04-T3 0.0336 0.0012 31 0.0255 0.0010 29 2.01E−06 99^(b)ME00572-04- -04-T4 0.0388 0.0014 57 0.0267 0.0005 294 −9.39E−1199-01^(c) ME00572-04- -04-T4 0.0345 0.0011 41 0.0253 0.0018 19 3.93E−0599-02 ME00572-04- -04-T4 0.0433 0.0012 60 0.0267 0.0005 294 −3.33E−1699-03^(c) ME00572-04- -04-T4 0.0315 0.0010 44 0.0239 0.0012 16 3.22E−0699-04 ME00572-05 -05-T2 0.0362 0.0020 19 0.0219 0.0020 10 1.13E−05ME00572-05- -05-T3 0.0337 0.0011 57 0.0267 0.0005 294 6.21E−09 01^(c)ME00572-05- -05-T3 0.0320 0.0014 39 0.0232 0.0020 17 3.59E−04 02ME00572-05- -05-T3 0.0379 0.0011 59 0.0267 0.0005 294 −9.39E−11 03^(c)ME00572-05- -05-T3 0.0344 0.0020 37 0.0268 0.0022 19 7.26E−03 04^(a)Transgenic seedlings were compared to internal non-transgenicsegregants within an event unless otherwise indicated. ^(b)These eventswere sown twice. The first time was to identify ME00572 as a Hit. Theywere repeated the second time with the next generation to identifyME00572 as a Lead. ^(c)These events did not segregate non-transgenicseedlings and were compared to pooled non- transgenics for the line.

Plants from Events-01, -03, -04 and -05 which are hemizygous orhomozygous for clone 13209 do not show any negative phenotypes understandard conditions. Events-01, -03, -04 and -05 of ME00572 were testedfor negative phenotypes compared to the empty vector control SR00559.There was no detectable reduction in germination rate, the plantsappeared wild-type in all instances, and there was no statisticaldifferences between experimentals and controls for days to flowering,rosette area 7 days post-bolting, and fertility (silique number and seedfill).

Example 7 Determination of Functional Homologs by Reciprocal BLAST

A candidate sequence was considered a functional homolog of a referencesequence if the candidate and reference sequences encoded proteinshaving a similar function and/or activity. A process known as ReciprocalBLAST (Rivera et al., Proc. Natl. Acad. Sci. USA, 95:6239-6244 (1998))was used to identify potential functional homolog sequences fromdatabases consisting of all available public and proprietary peptidesequences, including NR from NCBI and peptide translations from Ceresclones.

Before starting a Reciprocal BLAST process, a specific referencepolypeptide was searched against all peptides from its source speciesusing BLAST in order to identify polypeptides having BLAST sequenceidentity of 80% or greater to the reference polypeptide and an alignmentlength of 85% or greater along the shorter sequence in the alignment.The reference polypeptide and any of the aforementioned identifiedpolypeptides were designated as a cluster.

The BLASTP version 2.0 program from Washington University at SaintLouis, Mo., USA was used to determine BLAST sequence identity andE-value. The BLASTP version 2.0 program includes the followingparameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3)the -postsw option. The BLAST sequence identity was calculated based onthe alignment of the first BLAST HSP (High-scoring Segment Pairs) of theidentified potential functional homolog sequence with a specificreference polypeptide. The number of identically matched residues in theBLAST HSP alignment was divided by the HSP length, and then multipliedby 100 to get the BLAST sequence identity. The HSP length typicallyincluded gaps in the alignment, but in some cases gaps were excluded.

The main Reciprocal BLAST process consists of two rounds of BLASTsearches; forward search and reverse search. In the forward search step,a reference polypeptide sequence, “polypeptide A,” from source speciesSA was BLASTed against all protein sequences from a species of interest.Top hits were determined using an E-value cutoff of 10⁻⁵ and a sequenceidentity cutoff of 35%. Among the top hits, the sequence having thelowest E-value was designated as the best hit, and considered apotential functional homolog or ortholog. Any other top hit that had asequence identity of 80% or greater to the best hit or to the originalreference polypeptide was considered a potential functional homolog orortholog as well. This process was repeated for all species of interest.

In the reverse search round, the top hits identified in the forwardsearch from all species were BLASTed against all protein sequences fromthe source species SA. A top hit from the forward search that returned apolypeptide from the aforementioned cluster as its best hit was alsoconsidered as a potential functional homolog.

Functional homologs were identified by manual inspection of potentialfunctional homolog sequences. Representative functional homologs for SEQID NO: 2, 20, 74, 93, and 116, are shown in FIGS. 1-5, respectively.Additional exemplary homologs are correlated to certain Figures in theSequence Listing.

Example 8 Determination of Functional Homologs by Hidden Markov Models

Hidden Markov Models (HMMs) were generated by the program HMMER 2.3.2.To generate each HMM, the default HMMER 2.3.2 program parameters,configured for glocal alignments, were used.

An HMM was generated using the sequences shown in FIG. 1 as input. Thesesequences were fitted to the model and a representative HMM bit scorefor each sequence is shown in the Sequence Listing. Additional sequenceswere fitted to the model, and representative HMM bit scores for any suchadditional sequences are shown in the Sequence Listing. The resultsindicate that these additional sequences are functional homologs of SEQID NO: 2.

The procedure above was repeated and an HMM was generated for each groupof sequences shown in FIGS. 2, 3, 4, and 5 using the sequences shown ineach Figure as input for that HMM. A representative bit score for eachsequence is shown in the Sequence Listing. Additional sequences werefitted to certain HMMs, and representative HMM bit scores for suchadditional sequences are shown in the Sequence Listing. The resultsindicate that these additional sequences are functional homologs of thesequences used to generate that HMM.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of producing a plant and/or plant tissue, said methodcomprising growing a plant cell comprising an exogenous nucleic acid,said exogenous nucleic acid comprising a regulatory region operablylinked to a nucleotide sequence encoding a polypeptide, wherein the HMMbit score of the amino acid sequence of said polypeptide is greater thanabout 130, said HMM based on the amino acid sequences depicted in one ofFIG. 1, 2, 3, 4, or 5 and wherein said plant and/or plant tissue has adifference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise said nucleic acid.
 2. The method of claim 1, wherein the HMMbit score of the amino acid is greater than about 180 based on the aminoacid sequences depicted in FIG.
 2. 3. The method of claim 1, wherein theHMM bit score of the amino acid is greater than about 650 based on theamino acid sequences depicted in FIG.
 3. 4. The method of claim 1,wherein the HMM bit score of the amino acid is greater than about 310,the HMM is based on the amino acid sequences depicted in FIG. 4, andwherein the polypeptide is about 150 to 170 amino acids in length.
 5. Amethod of producing a plant and/or plant tissue, said method comprisinggrowing a plant cell comprising an exogenous nucleic acid, saidexogenous nucleic acid comprising a regulatory region operably linked toa nucleotide sequence encoding a polypeptide having 80 percent orgreater sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 15, 17, 20, 20,22, 24, 26, 28, 29, 30, 32, 34, 36, 38, 40, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 56, 58, 59, 60, 61, 62, 63, 64, 65, 68, 69, 71,74, 76, 77, 79, 81, 82, 83, 85, 86, 88, 90, 93, 95, 96, 98, 100, 101,102, 104, 106, 107, 108, 110, 112, 114, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 134, 136, 137, 138,139, 141, 143, 170, 172, 173, 175, 176, 178, 180, 181, 182, 183, 185,186, 187, 189, 190, 192, 193, 194, 195, 196, 198, 199, 201, 203, 204,205, 206, 207, 208, 210, 211, 212, 213, 214, 216, 218, 219, 220, 221,223, 163, 164, 165, 167, 169, 157, 158, 159, 160, 161, 225, 226, 227,229, 231, 233, 235, 237, 239, 241, 242, 244, 246, 247, 249, 250, 252,254, 255, 256, 258, 259, and 260, wherein a plant and/or plant tissueproduced from said plant cell has a difference in the level of coldtolerance as compared to the corresponding level of cold tolerance of acontrol plant that does not comprise said nucleic acid.
 6. The method ofclaim 1 or 5, wherein the polypeptide comprises a B-box zinc fingerdomain having 60 percent or greater sequence identity to the B-box zincfinger domain of residues 56 to 103 of SEQ ID NO: 20 and a CCT motifhaving 60 percent or greater sequence identity to the CCT motif ofresidues 285 to 329 of SEQ ID NO:
 20. 7. The method of claim 1 or 5,wherein the polypeptide comprises a short chain dehydrogenase domainhaving 60 percent or greater sequence identity to the short chaindehydrogenase domain of residues 38 to 173 of SEQ ID NO:
 74. 8. Themethod of claim 1 or 5, wherein the polypeptide comprises a B3 DNAbinding domain having 60 percent or greater sequence identity to the B3DNA binding domain of residues 163 to 268 of SEQ ID NO: 112 and a auxinresponse factor domain having 60 percent or greater sequence identity tothe auxin response factor domain of residues 290 to 372 of SEQ ID NO:112.
 9. The method of claim 5, wherein the HMM bit score of the aminoacid sequence of said polypeptide is greater than about 130, said HMMbased on the amino acid sequences depicted in one of FIG. 1, 2, 3, 4, or5.
 10. The method of claim 1 or 5, wherein said polypeptide is selectedfrom the group consisting of SEQ ID NO: 2, 20, and
 93. 11. A method ofproducing a plant according to claim 1, wherein said method comprisesgrowing a plant cell comprising an exogenous nucleic acid, saidexogenous nucleic acid comprising a regulatory region operably linked toa nucleotide sequence or its complement having 80 percent or greatersequence identity of a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 14, 16, 18, 19, 21, 23, 25,27, 31, 33, 35, 37, 39, 41, 55, 57, 66, 66, 67, 67, 70, 72, 73, 75, 78,80, 84, 87, 89, 91, 92, 94, 97, 99, 103, 105, 109, 111, 113, 115, 129,133, 135, 140, 142, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 162, 166, 168, 171, 174, 177, 179, 184, 188, 191, 197,200, 202, 209, 215, 217, 222, 224, 228, 230, 232, 234, 236, 238, 240,243, 245, 248, 251, 253, and 257, or a fragment thereof, wherein a plantproduced from said plant cell has a difference in the level of coldtolerance as compared to the corresponding level of cold tolerance of acontrol plant that does not comprise said nucleic acid.
 12. A method ofmodulating the level of cold tolerance in a plant, said methodcomprising introducing into a plant cell an exogenous nucleic acid, saidexogenous nucleic acid comprising a regulatory region operably linked toa nucleotide sequence encoding a polypeptide, wherein the HMM bit scoreof the amino acid sequence of said polypeptide is greater than about130, said HMM based on the amino acid sequences depicted in one of FIG.1, 2, 3, 4, or 5, and wherein a plant produced from said plant cell hasa difference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise said exogenous nucleic acid.
 13. A method of modulating thelevel of cold tolerance in a plant according to claim 12 comprisingintroducing into a plant cell an exogenous nucleic acid, said exogenousnucleic acid comprising a regulatory region operably linked to anucleotide sequence encoding a polypeptide having 80 percent or greatersequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 15, 17, 20, 20, 22, 24,26, 28, 29, 30, 32, 34, 36, 38, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 56, 58, 59, 60, 61, 62, 63, 64, 65, 68, 69, 71, 74, 76,77, 79, 81, 82, 83, 85, 86, 88, 90, 93, 95, 96, 98, 100, 101, 102, 104,106, 107, 108, 110, 112, 114, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 130, 131, 132, 134, 136, 137, 138, 139, 141,143, 170, 172, 173, 175, 176, 178, 180, 181, 182, 183, 185, 186, 187,189, 190, 192, 193, 194, 195, 196, 198, 199, 201, 203, 204, 205, 206,207, 208, 210, 211, 212, 213, 214, 216, 218, 219, 220, 221, 223, 163,164, 165, 167, 169, 157, 158, 159, 160, 161, 225, 226, 227, 229, 231,233, 235, 237, 239, 241, 242, 244, 246, 247, 249, 250, 252, 254, 255,256, 258, 259, and 260, wherein a plant produced from said plant cellhas a difference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise said nucleic acid.
 14. A method of modulating the level of coldtolerance in a plant according to claim 12, comprising introducing intoa plant cell an exogenous nucleic acid, said exogenous nucleic acidcomprising a regulatory region operably linked to a nucleotide sequencehaving 80 percent or greater sequence identity to a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 14,16, 18, 19, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41, 55, 57, 66, 66, 67,67, 70, 72, 73, 75, 78, 80, 84, 87, 89, 91, 92, 94, 97, 99, 103, 105,109, 111, 113, 115, 129, 133, 135, 140, 142, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 162, 166, 168, 171, 174, 177,179, 184, 188, 191, 197, 200, 202, 209, 215, 217, 222, 224, 228, 230,232, 234, 236, 238, 240, 243, 245, 248, 251, 253, and 257, or a fragmentthereof, wherein a plant produced from said plant cell has a differencein the level of cold tolerance as compared to the corresponding level ofcold tolerance of a control plant that does not comprise said nucleicacid.
 15. A plant cell comprising an exogenous nucleic acid saidexogenous nucleic acid comprising a regulatory region operably linked toa nucleotide sequence encoding a polypeptide, wherein the HMM bit scoreof the amino acid sequence of said polypeptide is greater than about130, said HMM based on the amino acid sequences depicted in one of FIG.1, 2, 3, 4, or 5, and wherein a plant produced from said plant cell hasa difference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise said nucleic acid.
 16. The plant cell of claim 15, wherein thepolypeptide has 80 percent or greater sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8,10, 12, 13, 15, 17, 20, 20, 22, 24, 26, 28, 29, 30, 32, 34, 36, 38, 40,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 58, 59, 60, 61,62, 63, 64, 65, 68, 69, 71, 74, 76, 77, 79, 81, 82, 83, 85, 86, 88, 90,93, 95, 96, 98, 100, 101, 102, 104, 106, 107, 108, 110, 112, 114, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131,132, 134, 136, 137, 138, 139, 141, 143, 170, 172, 173, 175, 176, 178,180, 181, 182, 183, 185, 186, 187, 189, 190, 192, 193, 194, 195, 196,198, 199, 201, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214,216, 218, 219, 220, 221, 223, 163, 164, 165, 167, 169, 157, 158, 159,160, 161, 225, 226, 227, 229, 231, 233, 235, 237, 239, 241, 242, 244,246, 247, 249, 250, 252, 254, 255, 256, 258, 259, and
 260. 17. A plantcell according to claim 15, comprising an exogenous nucleic acid saidexogenous nucleic acid comprising a regulatory region operably linked toa nucleotide sequence encoding a polypeptide having 80 percent orgreater sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 15, 17, 20, 20,22, 24, 26, 28, 29, 30, 32, 34, 36, 38, 40, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 56, 58, 59, 60, 61, 62, 63, 64, 65, 68, 69, 71,74, 76, 77, 79, 81, 82, 83, 85, 86, 88, 90, 93, 95, 96, 98, 100, 101,102, 104, 106, 107, 108, 110, 112, 114, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 134, 136, 137, 138,139, 141, 143, 170, 172, 173, 175, 176, 178, 180, 181, 182, 183, 185,186, 187, 189, 190, 192, 193, 194, 195, 196, 198, 199, 201, 203, 204,205, 206, 207, 208, 210, 211, 212, 213, 214, 216, 218, 219, 220, 221,223, 163, 164, 165, 167, 169, 157, 158, 159, 160, 161, 225, 226, 227,229, 231, 233, 235, 237, 239, 241, 242, 244, 246, 247, 249, 250, 252,254, 255, 256, 258, 259, and 260, wherein a plant produced from saidplant cell has a difference in the level of cold tolerance as comparedto the corresponding level of cold tolerance of a control plant thatdoes not comprise said nucleic acid.
 18. A plant cell according to claim15, comprising an exogenous nucleic acid said exogenous nucleic acidcomprising a regulatory region operably linked to a nucleotide sequenceor its complement having 80 percent or greater sequence identity of anucleotide sequence selected from the group consisting of SEQ ID NO: 1,3, 5, 7, 9, 11, 14, 16, 18, 19, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41,55, 57, 66, 66, 67, 67, 70, 72, 73, 75, 78, 80, 84, 87, 89, 91, 92, 94,97, 99, 103, 105, 109, 111, 113, 115, 129, 133, 135, 140, 142, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 162, 166, 168,171, 174, 177, 179, 184, 188, 191, 197, 200, 202, 209, 215, 217, 222,224, 228, 230, 232, 234, 236, 238, 240, 243, 245, 248, 251, 253, and257, or a fragment thereof, wherein a plant produced from said plantcell has a difference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise said nucleic acid.
 19. A transgenic plant comprising the plantcell of any one of claims 15-18.
 20. The transgenic plant of claim 19,wherein said polypeptide is selected from the group consisting of SEQ IDNO: 2, 20, and
 93. 21. The transgenic plant of claim 19, wherein saidplant is a member of a species selected from the group consisting ofPanicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass),Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populusbalsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassicanapus (canola), Triticum aeslivum (wheat), Gossypium hirsutum (cotton),Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa(alfalfa), Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearlmillet).
 22. A seed product comprising embryonic tissue from atransgenic plant according to claim
 19. 23. An isolated nucleic acidcomprising a nucleotide sequence having 95% or greater sequence identityto the nucleotide sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 14,16, 21, 23, 25, 27, 33, 37, 39, 41, 55, 57, 70, 75, 80, 84, 87, 91, 92,97, 105, 113, 115, 129, 133, or
 140. 24. An isolated nucleic acidcomprising a nucleotide sequence encoding a polypeptide having 80% orgreater sequence identity to the amino acid sequence set forth in SEQ IDNO: 4, 6, 8, 10, 12, 22, 24, 26, 28, 36, 38, 40, 42, 71, 74, 85, 88, 93,105, 114, 116, 130, 134, 136, 141, or
 143. 25. A method of identifyingwhether a polymorphism is associated with variation in a trait, saidmethod comprising: a) determining whether one or more geneticpolymorphisms in a population of plants is associated with the locus fora polypeptide selected from the group consisting of the polypeptidesdepicted in FIGS. 1, 2, 3, 4, or 5 and functional homologs thereof; andb) measuring the correlation between variation in said trait in plantsof said population and the presence of said one or more geneticpolymorphisms in plants of said population, thereby identifying whetheror not said one or more genetic polymorphisms are associated withvariation in said trait.
 26. A method of making a plant line, saidmethod comprising: a) determining whether one or more geneticpolymorphisms in a population of plants is associated with the locus fora polypeptide selected from the group consisting of the polypeptidesdepicted in FIGS. 1, 2, 3, 4, or 5 and functional homologs thereof; b)identifying one or more plants in said population in which the presenceof at least one allele at said one or more genetic polymorphisms isassociated with variation in a trait; c) crossing each said one or moreidentified plants with itself or a different plant to produce seed; d)crossing at least one progeny plant grown from said seed with itself ora different plant; and e) repeating steps c) and d) for an additional0-5 generations to make said plant line, wherein said at least oneallele is present in said plant line.
 27. The method of claim 25 or 26,wherein said trait is the level of cold tolerance.
 28. The method ofclaim 25, wherein said population is a population of switchgrass,sorghum, sugar cane, or miscanthus plants.
 29. A plant cell comprisingan exogenous nucleic acid said exogenous nucleic acid comprising aregulatory region operably linked to a nucleotide sequence comprising atleast a fragment having 80 percent or greater sequence identity to anucleic acid sequence selected from the group consisting of residues 305to about 346 of SEQ ID NO: 111, residues 21 to about 62 of SEQ ID NO:66, residues 20 to about 61 of SEQ ID NO: 67, residues 21 to about 62 ofSEQ ID NO: 72, residues 21 to about 62 of SEQ ID NO: 73, residues 77 toabout 118 of SEQ ID NO: 144, residues 292 to about 313 of SEQ ID NO:145, residues 37 to about 78 of SEQ ID NO: 146, residues 56 to about 97of SEQ ID NO: 147, residues 37 to about 78 of SEQ ID NO: 148, residues45 to about 86 of SEQ ID NO: 149, residues 46 to about 98 of SEQ ID NO:150, residues 476 to about 497 of SEQ ID NO: 151, residues 21 to about62 of SEQ ID NO: 152, residues 21 to about 62 of SEQ ID NO: 153,residues 21 to about 62 of SEQ ID NO: 154, residues 21 to about 62 ofSEQ ID NO: 155, and residues 21 to about 62 of SEQ ID NO: 156, or theircomplement, wherein a plant produced from said plant cell has adifference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise said nucleic acid.
 30. A transgenic plant comprising the plantcell of claim
 29. 31. The transgenic plant of claim 30, wherein saidexogenous nucleic acid comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 66, 67, 72, 73, 111, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, and
 156. 32. A method ofproducing a plant, said method comprising introducing into a plant cellan exogenous nucleic acid, said exogenous nucleic acid comprising aregulatory region operably linked to a nucleotide sequence encoding atleast a fragment having 80 percent or greater sequence identity to anucleic acid sequence selected from the group consisting of residues 305to about 346 of SEQ ID NO: 111, residues 21 to about 62 of SEQ ID NO:66, residues 20 to about 61 of SEQ ID NO: 67, residues 21 to about 62 ofSEQ ID NO: 72, residues 21 to about 62 of SEQ ID NO: 73, residues 77 toabout 118 of SEQ ID NO: 144, residues 292 to about 313 of SEQ ID NO:145, residues 37 to about 78 of SEQ ID NO: 146, residues 56 to about 97of SEQ ID NO: 147, residues 37 to about 78 of SEQ ID NO: 148, residues45 to about 86 of SEQ ID NO: 149, residues 46 to about 98 of SEQ ID NO:150, residues 476 to about 497 of SEQ ID NO: 151, residues 21 to about62 of SEQ ID NO: 152, residues 21 to about 62 of SEQ ID NO: 153,residues 21 to about 62 of SEQ ID NO: 154, residues 21 to about 62 ofSEQ ID NO: 155, and residues 21 to about 62 of SEQ ID NO: 156, or theircomplement, wherein a plant produced from said plant cell has adifference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise said nucleic acid.
 33. The method of claim 32, whereinexpression of a target polypeptide is suppressed, said targetpolypeptide having 80 percent or greater sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NO: 112, 114,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130,131, 132, 134, 136, 137, 138, 139, 141, and
 143. 34. The method of claim33, wherein the HMM bit score of the amino acid sequence of saidpolypeptide is greater than about 790, said HMM based on the amino acidsequences depicted in FIG.
 5. 35. The method of claim 32, wherein thenucleotide sequence or its complement is complementary to RNAtranscribed from a gene encoding said target polypeptide.
 36. The methodof claim 32, wherein the nucleotide sequence comprises a microRNArecognition site having 80 percent or greater sequence identity to anucleic acid sequence selected from the group consisting of residues 109to about 129 of SEQ ID NO: 66, residues 114 to about 135 of SEQ ID NO:67, residues 119 to about 139 of SEQ ID NO: 72, residues 108 to about128 of SEQ ID NO: 73, residues 234 to about 254 of SEQ ID NO: 144,residues 135 to about 176 of SEQ ID NO: 145, residues 173 to about 189of SEQ ID NO: 147, residues 154 to about 170 of SEQ ID NO: 148, residues134 to about 157 of SEQ ID NO: 149, residues 154 to about 198 of SEQ IDNO: 150, residues 319 to about 360 of SEQ ID NO: 151, residues 121 toabout 141 of SEQ ID NO: 152, residues 120 to about 140 of SEQ ID NO:153, residues 121 to about 141 of SEQ ID NO: 154, residues 121 to about141 of SEQ ID NO: 155, residues 121 to about 141 of SEQ ID NO: 156, andresidues 462 to about 483 of SEQ ID NO:
 111. 37. A method of modulatingthe level of cold tolerance in a plant, said method comprisingintroducing into a plant cell an exogenous nucleic acid, said exogenousnucleic acid comprising a regulatory region operably linked to anucleotide sequence or its complement having 80 percent or greatersequence identity to a nucleic acid sequence selected from the groupconsisting of residues 305 to about 346 of SEQ ID NO: 111, residues 21to about 62 of SEQ ID NO: 66, residues 20 to about 61 of SEQ ID NO: 67,residues 21 to about 62 of SEQ ID NO: 72, residues 21 to about 62 of SEQID NO: 73, residues 77 to about 118 of SEQ ID NO: 144, residues 292 toabout 313 of SEQ ID NO: 145, residues 37 to about 78 of SEQ ID NO: 146,residues 56 to about 97 of SEQ ID NO: 147, residues 37 to about 78 ofSEQ ID NO: 148, residues 45 to about 86 of SEQ ID NO: 149, residues 46to about 98 of SEQ ID NO: 150, residues 476 to about 497 of SEQ ID NO:151, residues 21 to about 62 of SEQ ID NO: 152, residues 21 to about 62of SEQ ID NO: 153, residues 21 to about 62 of SEQ ID NO: 154, residues21 to about 62 of SEQ ID NO: 155, and residues 21 to about 62 of SEQ IDNO: 156, or a fragment thereof, wherein a plant produced from said plantcell has a difference in the level of cold tolerance as compared to thecorresponding level of cold tolerance of a control plant that does notcomprise said nucleic acid.